The evolution of new and reemerging historic virulent strains of respiratory viruses from animal reservoirs is a significant threat to human health. Inefficient human-to-human transmission of zoonotic strains may initially limit the spread of transmission, but an infection may be contracted by touching contaminated surfaces. Enveloped viruses are often susceptible to environmental stresses, but the human coronaviruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have recently caused increasing concern of contact transmission during outbreaks. We report here that pathogenic human coronavirus 229E remained infectious in a human lung cell culture model following at least 5 days of persistence on a range of common nonbiocidal surface materials, including polytetrafluoroethylene (Teflon; PTFE), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. We have shown previously that noroviruses are destroyed on copper alloy surfaces. In this new study, human coronavirus 229E was rapidly inactivated on a range of copper alloys (within a few minutes for simulated fingertip contamination) and Cu/Zn brasses were very effective at lower copper concentration. Exposure to copper destroyed the viral genomes and irreversibly affected virus morphology, including disintegration of envelope and dispersal of surface spikes. Cu(I) and Cu(II) moieties were responsible for the inactivation, which was enhanced by reactive oxygen species generation on alloy surfaces, resulting in even faster inactivation than was seen with nonenveloped viruses on copper. Consequently, copper alloy surfaces could be employed in communal areas and at any mass gatherings to help reduce transmission of respiratory viruses from contaminated surfaces and protect the public health.
We have reported previously that copper I and II ionic species, and superoxide but not Fenton reaction generated hydroxyl radicals, are important in the killing mechanism of pathogenic enterococci on copper surfaces. In this new work we determined if the mechanism was the same in non-pathogenic ancestral (K12) and laboratory (DH5α) strains, and a pathogenic strain (O157), of Escherichia coli. The pathogenic strain exhibited prolonged survival on stainless steel surfaces compared with the other E. coli strains but all died within 10 min on copper surfaces using a 'dry' inoculum protocol (with approximately 10(7) cfu cm(-2) ) to mimic dry touch contamination. We observed immediate cytoplasmic membrane depolarization, not seen with enterococci or methicillin resistant Staphylococcus aureus, and loss of outer membrane integrity, inhibition of respiration and in situ generation of reactive oxygen species on copper and copper alloy surfaces that did not occur on stainless steel. Chelation of copper (I) and (II) ionic species still had the most significant impact on bacterial survival but protection by d-mannitol suggests hydroxyl radicals are involved in the killing mechanism. We also observed a much slower rate of DNA destruction on copper surfaces compared with previous results for enterococci. This may be due to protection of the nucleic acid by the periplasm and the extensive cell aggregation that we observed on copper surfaces. Similar results were obtained for Salmonella species but partial quenching by d-mannitol suggests radicals other than hydroxyl may be involved. The results indicate that copper biocidal surfaces are effective for Gram-positive and Gram-negative bacteria but bacterial morphology affects the mechanism of toxicity. These surfaces could not only help to prevent infection spread but also prevent horizontal gene transmission which is responsible for the evolution of virulent toxin producing and antibiotic resistant bacteria.
The increasing incidence of nosocomial infections caused by glycopeptide-resistant enterococci is a global concern. Enterococcal species are also difficult to eradicate with existing cleaning regimens; they can survive for long periods on surfaces, thus contributing to cases of reinfection and spread of antibiotic-resistant strains. We have investigated the potential use of copper alloys as bactericidal surfaces. Clinical isolates of vancomycinresistant Enterococcus faecalis and Enterococcus faecium were inoculated onto copper alloy and stainless steel surfaces. Samples were assessed for the presence of viable cells by conventional culture, detection of actively respiring cells, and assessment of cell membrane integrity. Both species survived for up to several weeks on stainless steel. However, no viable cells were detected on any alloys following exposure for 1 h at an inoculum concentration of <10 4 CFU/cm 2 . Analysis of genomic and plasmid DNA from bacterial cells recovered from metal surfaces indicates substantial disintegration of the DNA following exposure to copper surfaces that is not evident in cells recovered from stainless steel. The DNA fragmentation is so extensive, and coupled with the rapid cell death which occurs on copper surfaces, that it suggests that mutation is less likely to occur. It is therefore highly unlikely that genetic information can be transferred to receptive organisms recontaminating the same area. A combination of effective cleaning regimens and contact surfaces containing copper could be useful not only to prevent the spread of viable pathogenic enterococci but also to mitigate against the occurrence of potential resistance to copper, biocides, or antibiotics and the spread of genetic determinants of resistance to other species.
Contaminated touch surfaces have been implicated in the spread of hospital-acquired infections, and the use of biocidal surfaces could help to reduce this cross-contamination. In a previous study we reported the death of aqueous inocula of pathogenic Enterococcus faecalis or Enterococcus faecium isolates, simulating fomite surface contamination, in 1 h on copper alloys, compared to survival for months on stainless steel. In our current study we observed an even faster kill of over a 6-log reduction of viable enterococci in less than 10 min on copper alloys with a "dry" inoculum equivalent to touch contamination. We investigated the effect of copper(I) and copper(II) chelation and the quenching of reactive oxygen species on cell viability assessed by culture and their effects on genomic DNA, membrane potential, and respiration in situ on metal surfaces. We propose that copper surface toxicity for enterococci involves the direct or indirect action of released copper ionic species and the generation of superoxide, resulting in arrested respiration and DNA breakdown as the first stages of cell death. The generation of hydroxyl radicals by the Fenton reaction does not appear to be the dominant instrument of DNA damage. The bacterial membrane potential is unaffected in the early stages of wet and dry surface contact, suggesting that the membrane is not compromised until after cell death. These results also highlight the importance of correct surface cleaning protocols to perpetuate copper ion release and prevent the chelation of ions by contaminants, which could reduce the efficacy of the surface.
Noroviruses (family Caliciviridae) are the primary cause of viral gastroenteritis worldwide. The virus is highly infectious and touching contaminated surfaces can contribute to infection spread. Although the virus was identified over 40 years ago the lack of methods to assess infectivity has hampered the study of the human pathogen. Recently the murine virus, MNV-1, has successfully been used as a close surrogate. Copper alloys have previously been shown to be effective antimicrobial surfaces against a range of bacteria and fungi. We now report rapid inactivation of murine norovirus on alloys, containing over 60% copper, at room temperature but no reduction of infectivity on stainless steel dry surfaces in simulated wet fomite and dry touch contamination. The rate of inactivation was initially very rapid and proportional to copper content of alloy tested. Viral inactivation was not as rapid on brass as previously observed for bacteria but copper-nickel alloy was very effective. The use of chelators and quenchers of reactive oxygen species (ROS) determined that Cu(II) and especially Cu(I) ions are still the primary effectors of toxicity but quenching superoxide and hydroxyl radicals did not confer protection. This suggests Fenton generation of ROS is not important for the inactivation mechanism. One of the targets of copper toxicity was the viral genome and a reduced copy number of the gene for a viral encoded protein, VPg (viral-protein-genome-linked), which is essential for infectivity, was observed following contact with copper and brass dry surfaces. The use of antimicrobial surfaces containing copper in high risk closed environments such as cruise ships and care facilities could help to reduce the spread of this highly infectious and costly pathogen.
Horizontal gene transfer (HGT) is largely responsible for increasing the incidence of antibiotic-resistant infections worldwide. While studies have focused on HGT in vivo, this work investigates whether the ability of pathogens to persist in the environment, particularly on touch surfaces, may also play an important role. Escherichia coli, virulent clone ST131, and Klebsiella pneumoniae harboring extended-spectrum-β-lactamase (ESBL) blaCTX-M-15 and metallo-β-lactamase blaNDM-1, respectively, exhibited prolonged survival on stainless steel, with approximately 104 viable cells remaining from an inoculum of 107 CFU per cm2 after 1 month at 21°C. HGT of bla to an antibiotic-sensitive but azide-resistant recipient E. coli strain occurred on stainless steel dry touch surfaces and in suspension but not on dry copper. The conjugation frequency was approximately 10 to 50 times greater and occurred immediately, and resulting transconjugants were more stable with ESBL E. coli as the donor cell than with K. pneumoniae, but blaNDM-1 transfer increased with time. Transconjugants also exhibited the same resistance profile as the donor, suggesting multiple gene transfer. Rapid death, inhibition of respiration, and destruction of genomic and plasmid DNA of both pathogens occurred on copper alloys accompanied by a reduction in bla copy number. Naked E. coli DNA degraded on copper at 21°C and 37°C but slowly at 4°C, suggesting a direct role for the metal. Persistence of viable pathogenic bacteria on touch surfaces may not only increase the risk of infection transmission but may also contribute to the spread of antibiotic resistance by HGT. The use of copper alloys as antimicrobial touch surfaces may help reduce infection and HGT.
Norovirus is one of the most common causes of acute viral gastroenteritis. The virus is spread via the fecal-oral route, most commonly from infected food and water, but several outbreaks have originated from contamination of surfaces with infectious virus. In this study, a close surrogate of human norovirus causing gastrointestinal disease in mice, murine norovirus type 1 (MNV-1), retained infectivity for more than 2 weeks following contact with a range of surface materials, including Teflon (polytetrafluoroethylene [PTFE]), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. Persistence was slightly prolonged on ceramic surfaces. A previous study in our laboratory observed that dry copper and copper alloy surfaces rapidly inactivated MNV-1 and destroyed the viral genome. In this new study, we have observed that a relatively small change in the percentage of copper, between 70 and 80% in copper nickels and 60 and 70% in brasses, had a significant influence on the ability of the alloy to inactivate norovirus. Nickel alone did not affect virus, but zinc did have some antiviral effect, which was synergistic with copper and resulted in an increased efficacy of brasses with lower percentages of copper. Electron microscopy of purified MNV-1 that had been exposed to copper and stainless steel surfaces suggested that a massive breakdown of the viral capsid had occurred on copper. In addition, MNV-1 that had been exposed to copper and treated with RNase demonstrated a reduction in viral gene copy number. This suggests that capsid integrity is compromised upon contact with copper, allowing copper ion access to the viral genome. N oroviruses are responsible for approximately half of all cases of gastroenteritis worldwide. Their low infectious dose, ability to persist in an infectious state in the environment, and resistance to many commonly used cleaning agents have led to many disease outbreaks that have proved very difficult to contain (1, 2). The virus is spread directly via the fecal-oral route but also from touching contaminated surfaces, which has recently been found to be more significant than originally thought in the spread of many diseases (3). Ineffective cleaning agents may leave on surfaces residual virus particles which can initiate an infection (4). Norovirus disease is usually self-limiting, with symptoms lasting a few days, but can be more serious in severely ill or immunocompromised individuals, especially if the causative agent is one of the emerging recombinant strains, including GII.g/GII.12, which appeared in 2008 and has enhanced virulence and severity of clinical symptoms. Asymptomatic carriage and extended virus shedding also increase the risk of transmission (5, 6). A recent study of a large waterborne outbreak in Nokia, Finland, also observed that norovirus exposure may result in long-term health effects, which can persist for 15 months after the initial infection (7). This may mean that the considerable public health costs incurred in initial outbreaks, estimate...
The pandemic of hospital-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has declined, but the evolution of strains with enhanced virulence and toxins and the increase of community-associated infections are still a threat. In previous studies, 10 7 MRSA bacteria applied as simulated droplet contamination were killed on copper and brass surfaces within 90 min. However, contamination of surfaces is often via finger tips and dries rapidly, and it may be overlooked by cleaning regimes (unlike visible droplets). In this new study, a 5-log reduction of a hardy epidemic strain of MRSA (epidemic methicillin-resistant S. aureus 16 [EMRSA-16]) was observed following 10 min of contact with copper, and a 4-log reduction was observed on copper nickel and cartridge brass alloys in 15 min. A methicillin-sensitive S. aureus (MSSA) strain from an osteomyelitis patient was killed on copper surfaces in 15 min, and 4-log and 3-log reductions occurred within 20 min of contact with copper nickel and cartridge brass, respectively. Bacterial respiration was compromised on copper surfaces, and superoxide was generated as part of the killing mechanism. In addition, destruction of genomic DNA occurs on copper and brass surfaces, allaying concerns about horizontal gene transfer and copper resistance. Incorporation of copper alloy biocidal surfaces may help to reduce the spread of this dangerous pathogen. Intrinsic penicillin resistance and acquisition of resistance to methicillin in the 1980s by Staphylococcus aureus led to a pandemic of infections worldwide. Initially, the majority of infections were contracted in health care environments, but incorporation of measures to control the spread, including preadmission screening, decolonization, improved disinfection, and antibiotic treatment, have stemmed the tide (1). The increased use of antibiotics required for the epidemic of infections caused by Gram-positive pathogens has allowed the evolution of multidrug-resistant Gram-negative pathogens, effectively transforming some commensal gut bacteria into potential killers. However, new strains of S. aureus that have acquired further virulence factors and toxins or that have adapted to a specific environment, for example, an increased ability to cause bacteremia (2), are still a considerable threat. There is now widespread community-associated methicillin-resistant S. aureus (CA-MRSA), and infections can spread within households, day care centers, and schools (3). In addition, Giuffre et al. observed an increasing incidence of MRSA in neonates (4). The ability of some strains of MRSA to revert to methicillin-susceptible isolates, particularly in skin and soft tissue infections, has been observed (5).Colonization with MRSA increases the risk of MRSA infection, particularly following illness, surgical procedures, and treatment with immunosuppressive drugs. Colonization and/or infection may also occur from touching contaminated surfaces. In the community, a recent study observed that 58% and 82% of surfaces in 19 f...
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