Legionella colonization and 3D spatial location within a Pseudomonas biofilm.

Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire's Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.

Impact of different hand-drying methods on surrounding environment: aerosolization of virus and bacteria, and transfer to surfaces.

BACKGROUND: In recent years, hand drying has been highlighted as a key step in appropriate hand hygiene, as moisture on hands can increase the transfer of micro-organisms from hands to surfaces and vice versa. AIM: To understand bacterial and viral aerosolization following hand drying, and study the transfer of micro-organisms from hands to surfaces after drying using different methods. METHODS: Groups of five volunteers had their hands pre-washed with soap, rinsed and dried, then inoculated with a concentrated mixture of Pseudomonas fluorescens and MS2 bacteriophage. Volunteers entered an empty washroom, one at a time, and rinsed their hands with water or washed their hands with soap prior to drying with a jet dryer or paper towels. Each volunteer applied one hand successively to various surfaces, while their other hand was sampled using the glove juice method. Both residual bacteria and viruses were quantified from the washroom air, surface swabs and hand samples. FINDINGS: P. fluorescens and MS2 bacteriophages were rarely aerosolized while drying hands for any of the drying methods studied. Results also showed limited, and similar, transfer of both micro-organisms studied on to surfaces for all drying methods. CONCLUSION: The use of jet dryers or paper towels produces low levels of aerosolization when drying hands in a washroom. Similarly, all drying methods result in low transfer to surfaces. While the coronavirus disease 2019 pandemic raised concerns regarding public washrooms, this study shows that all methods tested are hygienic solutions for dry washed hands.

Modelling hospital disinfectant against multi-drug-resistant dry surface biofilms grown under artificial human sweat.

BACKGROUND: Dry surface biofilms (DSBs) have been found abundantly across hospital surfaces within intensive care units and may explain how nosocomial pathogens can remain virulent and persist on surfaces for extended periods. Testing standards governing the performance of disinfectant products employ planktonic models under routine growth conditions, which are known to be less tolerant than their biofilm counterpart. AIM: To evaluate biofilm models cultured under artificial human sweat (AHS), a source of nutrient expected on touch surfaces, to assess the antimicrobial performance of common cleaning agents, including a quaternary ammonium, hydrogen peroxide and active chlorine. METHODS: Five single-species biofilms, using pathogenic bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis, were generated on stainless-steel substrates using a sedimentation protocol under both AHS and nutrient-rich conditions for a direct comparison of phenotypic tolerance. The biofilm models were grown over five days followed by desiccation cycles, before being submerged into the disinfectant solutions for up to 25 min. Epifluorescence (EF) microscopy using LIVE/DEAD™ stain was used to visualize microcolony viability. FINDINGS: The results revealed biofilms cultured under AHS exhibited a greater antimicrobial tolerance and reduced speed of kill for all cleaning agents compared with the routine media; an average reduction of 72.4% vs 96.9%, respectively. EF microscopy revealed traces of viable bacteria across all coupons after disinfection indicating a potential opportunity for regrowth and recontamination. CONCLUSION: The notable difference in biocidal performance between the two growth conditions highlights potential pitfalls within current antimicrobial test standards, and the importance of accurate representation of the microbial challenge.

Identification of diverse antibiotic resistant bacteria in agricultural soil with H218O stable isotope probing combined with high-throughput sequencing.

BACKGROUND: We aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled "heavy" and the unlabelled "light" SIP fractions were sequenced. RESULTS: An increase of the 16S rRNA copy numbers in the "heavy" fractions of the treatments with 18O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) were highly abundant after two days of incubation with antibiotics. Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. In comparison, only two ARGs from the unbinned-assembled light fractions were identified. CONCLUSIONS: The results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.

Evaluation of cold atmospheric plasma for the decontamination of flexible endoscopes.

BACKGROUND: Despite adherence to standard protocols, residues including live micro-organisms may remain on the various surfaces of reprocessed flexible endoscopes. Prions are infectious proteins that are notoriously difficult to eliminate. AIM: To test the potential of cold atmospheric plasma (CAP) for the decontamination of various surfaces of flexible endoscopes, measuring total proteins and prion residual infectivity as indicators of efficacy. METHODS: New PTFE endoscope channels and metal test surfaces spiked with test soil or prion-infected tissues were treated using different CAP-generating prototypes. Surfaces were examined for the presence of residues using very sensitive fluorescence epimicroscopy. Prion residual infectivity was determined using the wire implant animal model and a more sensitive cell infectivity assay. FINDINGS: A CAP jet applied perpendicularly at close range on flat test surfaces removed soil within 3 min, but left microscopic residues and failed to eliminate prion infectivity according to the wire implant animal assay. The longitudinal gas flow from CAP prototypes developed for the treatment of long channels led to the displacement and sedimentation of residual soil towards the distal end, when applied alone. Observations of the plasma inside glass tubes showed temporal and spatial heterogeneity within a limited range. After the standard enzymatic manual pre-wash, 'CAP-activated' gas effluents prevented prion transmission from treated endoscope channels according to the prion infectivity cell assay. CONCLUSION: CAP shows promising results as a final step for decontamination of surgical surfaces. Optimizing CAP delivery could further enhance CAP efficacy, offering a safe, chemical-free alternative for the reprocessing of all luminal flexible endoscope surfaces.

Sensitive microscopic quantification of surface-bound prion infectivity for the assessment of surgical instrument decontamination procedures.

BACKGROUND: Pathogenic prions (PrPSc) are amyloid-rich hydrophobic proteins which bind avidly to surgical surfaces and represent some of the most difficult targets during the reprocessing of reusable surgical instruments. In-vitro methods to amplify and detect the presence of otherwise undetectable prion contamination are available, but they do not measure associated infectivity. Most of these methods rely on the use of proteinase K, however this can lead to the loss of a substantial portion of PrPSc, potentially producing false negatives. AIM: To develop a sensitive in-situ method without proteinase treatment for the dynamic quantification of amyloid accumulation in N2a #58 cells following 22L-prion infection from infected tissues and spiked stainless-steel surfaces. METHODS: We spiked cultures of N2a #58 cells with the 22L prion strain in solution or dried on stainless-steel wires and directly measured the accumulation of prion amyloid aggregates over several passages using highly sensitive fluorescence microscopy. FINDINGS: We demonstrated a 10-log dynamic range using our method to test residual prion infectivity, that was validated to show variable decontamination efficacy against prions from commercially available cleaning chemistries. CONCLUSIONS: The new cell-based infectivity method presented here avoids partial or possibly total proteinase K digestion of PrPSc in samples for greater sensitivity, in addition to low cost, no ethical concerns, and adaptability to detect different prion strains. This method can be used to test cleaning chemistries' efficacy with greater sensitivity than measuring total residual proteins, which may not correlate with residual prion infectivity.

Evaluating the environmental microbiota across four National Health Service hospitals within England.

Hospital surfaces contaminated with microbial soiling, such as dry surface biofilms (DSBs), can act as a reservoir for pathogenic micro-organisms, and inhibit their detection and removal during routine cleaning. Studies have recognized that such increases in bioburden can hinder the impact of disinfectants and mask the detection of potential pathogens. Cleanliness within healthcare settings is often determined through routine culture-based analysis, whereby surfaces that exhibit >2.5 colony-forming units (CFU) per cm2 pose a risk to patient health; therefore, any underestimation could have detrimental effects. This study quantified microbial growth on high-touch surfaces in four hospitals in England over 19 months. This was achieved using environmental swabs to sample a variety of surfaces within close proximity of the patient, and plating these on to non-specific low nutrient detection agar. The presence of DSBs on surfaces physically removed from the environment was confirmed using real-time imaging through episcopic differential interference contrast microscopy combined with epifluorescence. Approximately two-thirds of surfaces tested exceeded the limit for cleanliness (median 2230 CFU/cm2), whilst 83% of surfaces imaged with BacLight LIVE/DEAD staining confirmed traces of biofilm. Differences in infection control methods, such as choice of surface disinfectants and cleaning personnel, were not reflected in the microbial variation observed and resulting risk to patients. This highlights a potential limitation in the effectiveness of the current standards for all hospital cleaning, and further development using representative clinical data is required to overcome this limitation.

Artificial Human Sweat as a Novel Growth Condition for Clinically Relevant Pathogens on Hospital Surfaces.

The emergence of biofilms on dry hospital surfaces has led to the development of numerous models designed to challenge the efficacious properties of common antimicrobial agents used in cleaning. This is in spite of limited research defining how dry surfaces are able to facilitate biofilm growth and formation in such desiccating and nutrient-deprived environments. While it is well established that the phenotypical response of biofilms is dependent on the conditions in which they are formed, most models incorporate a nutrient-enriched, hydrated environment dissimilar to the clinical setting. In this study, we piloted a novel culture medium, artificial human sweat (AHS), which is perceived to be more indicative of the nutrient sources available on hospital surfaces, particularly those in close proximity to patients. AHS was capable of sustaining the proliferation of four clinically relevant multidrug-resistant pathogens (Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) and achieved biofilm formation at concentration levels equivalent to those found in situ (average, 6.00 log10 CFU/cm2) with similar visual characteristics upon microscopy. The AHS model presented here could be used for downstream applications, including efficacy testing of hospital cleaning products, due to its resemblance to clinical biofilms on dry surfaces. This may contribute to a better understanding of the true impact these products have on surface hygiene. IMPORTANCE Precise modeling of dry surface biofilms in hospitals is critical for understanding their role in hospital-acquired infection transmission and surface contamination. Using a representative culture condition which includes a nutrient source is key to developing a phenotypically accurate biofilm community. This will enable accurate laboratory testing of cleaning products and their efficacy against dry surface biofilms.

Synergism versus Additivity: Defining the Interactions between Common Disinfectants.

Many of the most common disinfectant and sanitizer products are formulations of multiple antimicrobial compounds. Products claiming to contain synergistic formulations are common, although there is often little supporting evidence. The antimicrobial interactions of all pairwise combinations of common disinfectants (benzalkonium chloride, didecyldimethylammonium chloride, polyhexamethylene biguanide, chlorocresol, and bronopol) were classified via checkerboard assay and validated by time-kill analyses. Combinations were tested against Acinetobacter baumannii NCTC 12156, Enterococcus faecalis NCTC 13379, Klebsiella pneumoniae NCTC 13443, and Staphylococcus aureus NCTC 13143. Synergistic interactions were identified only for the combinations of chlorocresol with benzalkonium chloride and chlorocresol with polyhexamethylene biguanide. Synergism was not ubiquitously demonstrated against all species tested and was on the borderline of the synergism threshold. These data demonstrate that synergism between disinfectants is uncommon and circumstantial. Most of the antimicrobial interactions tested were characterized as additive. We suggest that this is due to the broad, nonspecific mechanisms associated with disinfectants not providing an opportunity for the combined activities of these compounds to exceed the sum of their parts. IMPORTANCE The scarcity of observed synergistic interactions suggests that in the case of many disinfectant-based products, combined mechanisms of interaction may be being misinterpreted. We emphasize the need to correctly differentiate between additivity and synergism in antimicrobial formulations, as inappropriate classification may lead to unnecessary issues in the event of regulatory changes. Furthermore, we question the need to focus on synergism and disregard additivity when considering combinations of disinfectants, as the benefits that synergistic interactions provide are not necessarily relevant to the application of the final product.

Biofilm Development on Urinary Catheters Promotes the Appearance of Viable but Nonculturable Bacteria.

Catheter-associated urinary tract infections have serious consequences, for both patients and health care resources. Much work has been carried out to develop an antimicrobial catheter. Although such developments have shown promise under laboratory conditions, none have demonstrated a clear advantage in clinical trials. Using a range of microbiological and advanced microscopy techniques, a detailed laboratory study comparing biofilm development on silicone, hydrogel latex, and silver alloy-coated hydrogel latex catheters was carried out. Biofilm development by Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis on three commercially available catheters was tracked over time. Samples were examined with episcopic differential interference contrast (EDIC) microscopy, culture analysis, and staining techniques to quantify viable but nonculturable (VBNC) bacteria. Both qualitative and quantitative assessments found biofilms to develop rapidly on all three materials. EDIC microscopy revealed the rough surface topography of the materials. Differences between culture counts and quantification of total and dead cells demonstrated the presence of VBNC populations, where bacteria retain viability but are not metabolically active. The use of nonculture-based techniques showed the development of widespread VBNC populations. These VBNC populations were more evident on silver alloy-coated hydrogel latex catheters, indicating a bacteriostatic effect at best. The laboratory tests reported here, which detect VBNC bacteria, allow more rigorous assessment of antimicrobial catheters, explaining why there is often minimal benefit to patients.IMPORTANCE Several antimicrobial urinary catheter materials have been developed, but, although laboratory studies may show a benefit, none have significantly improved clinical outcomes. The use of poorly designed laboratory testing and lack of consideration of the impact of VBNC populations may be responsible. While the presence of VBNC populations is becoming more widely reported, there remains a lack of understanding of the clinical impact or influence of exposure to antimicrobial products. This is the first study to investigate the impact of antimicrobial surface materials and the appearance of VBNC populations. This demonstrates how improved testing is needed before clinical trials are initiated.

Improved surveillance of surgical instruments reprocessing following the variant Creutzfeldt-Jakob disease crisis in England: findings from a three-year survey.

BACKGROUND: Sensitive, direct protein-detection methods are now recommended for the inspection of reprocessed reusable surgical instruments in England to reduce the risk of prion transmission. AIM: To implement an established, highly sensitive method to quantify proteinaceous residues on reprocessed instruments in a Sterile Services Department (SSD) and evaluate its potential impact on service provision. METHODS: We introduced highly sensitive epifluorescence (EDIC/EF) microscopy in a large SSD. Over three years, we periodically tested two models of washer disinfector using stainless-steel tokens spiked with mouse brain homogenate or Browne test soil for comparison. We also obtained data and feedback from staff who had been using EDIC/EF to examine almost 3000 reprocessed instruments. FINDINGS: All reprocessed test surfaces harboured residual contamination (up to 258.4 ng from 1-µg spikes). Proximity between surfaces affected decontamination efficacy and allowed cross-contamination. Up to 50 ng de novo proteinaceous contamination was deposited on control surfaces after a single automated washer disinfector (AWD) cycle. The test soil behaved differently than real tissue contamination. SSD staff observed proteinaceous residues on most reprocessed instruments using EDIC/EF, which can detect far smaller amounts than the currently accepted national threshold of 5 µg per side. CONCLUSIONS: Implementing recent national guidelines to address the prions concern proved an eye-opener. Microscopic levels of proteins remain on many reprocessed instruments. The impact most of these residues, potentially including prions, may have on subsequent patients after sterilization remains debatable. Improving surveillance capability in SSDs can support decision making and raise the standards of surgical instruments reprocessing.

A cold water, ultrasonically activated stream efficiently removes proteins and prion-associated amyloid from surgical stainless steel.

BACKGROUND: Sterile service department decontamination procedures for surgical instruments struggle to demonstrate efficient removal of the hardiest infectious contaminants, such as prion proteins. A recently designed novel system, which uses a low pressure ultrasonically activated, cold water stream, has previously demonstrated efficient hard surface cleaning of several biological contaminants. AIM: To test the efficacy of an ultrasonically activated stream for the removal of tissue proteins, including prion-associated amyloid, from surgical stainless steel surfaces. METHODS: Test surfaces were contaminated with 22L, ME7 or 263K prion-infected brain homogenates. The surfaces were treated with the ultrasonically activated water stream for contact times of 5 and 10 s. Residual proteinaceous and amyloid contamination were quantified using sensitive microscopic analysis, and immunoblotting was used to characterize the eluted prion residues before and after treatment with the ultrasonically activated stream. FINDINGS: Efficient removal of the different prion strains from the surgical stainless steel surfaces was observed, and reduced levels of protease-susceptible and -resistant prion protein was detected in recovered supernatant. CONCLUSION: This study demonstrated that an ultrasonically activated stream has the potential to be a cost-effective solution to improve current decontamination practices and has the potential to reduce hospital-acquired infections.

An effective evidence-based cleaning method for the safe reuse of intermittent urinary catheters: In vitro testing.

AIMS: To determine a safe bactericidal cleaning method that does not damage urethral catheters used for intermittent catheterization. In some countries, single-use catheters are the norm; in others, the reuse of catheters is common depending on health insurance, personal preference, or individual concerns about the environment. However, no recent study of cleaning methods has been published to provide evidence for the safe reuse of catheters. METHODS: Using advanced microbiological methods, a laboratory study of eight cleaning methods was conducted. Sections of uncoated polyvinylchloride (PVC) catheters were exposed to bacterial uropathogens in physiologically correct artificial urine media then tested with a range of heat, chemical, and mechanical cleaning methods. Analysis of culturable and viable but nonculturable (VBNC) bacteria was done and direct microscopy was used. Descriptive statistics were used to compare values. RESULTS: Heat treatments, although effective, resulted in catheter surface breakdown and damage. Ultrasonic cleaning and vinegar showed evidence of VBNC populations indicating the methods were bacteriostatic. Detergent and water wash followed by immersion in a commercially available 0.6% sodium hypochlorite solution and 16.5% sodium chloride (diluted Milton) gave consistent bactericidal results and no visible catheter damage. CONCLUSIONS: Combined mechanical and chemical treatment of a detergent and water wash followed by immersion in diluted Milton (the "Milton Method") provided consistent and effective cleaning of uncoated PVC catheters, showing bactericidal action for all uropathogens tested after repeated exposure. If found safe in clinical testing, this method could increase the reuse of catheters, reduce plastic waste in the environment, reduce cost, and increase patient choice.

Modelling vaporised hydrogen peroxide efficacy against mono-species biofilms.

This pilot study investigates a novel approach towards efficacy testing of antimicrobial cleaning agents; focusing primarily on hydrogen peroxide vapour (HPV). Contaminated surfaces are recognised modes of pathogen transmission within healthcare environments and increase the risk of pathogen acquisition in newly admitted patients. Studies have shown these pathogens can survive on surfaces for extended periods of time in spite of cleaning. This resilience is characteristic of biofilm formation and recent publications have identified their presence in hospitals. In this study, biofilm models comprised of multidrug-resistant organisms (MDROs) were generated using a drip flow reactor and exposed to HPV decontamination. The MDROs included Acinetobacter baumannii, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Upon exposure, samples were periodically removed and enumerated to generate kill curves for each species. Consequently revealing any inherent resistances; such as catalase-producing organisms which expressed reduced susceptibility. Epifluorescence microscopy revealed an abundance of viable and non-viable microcolonies before and after decontamination, respectively. Greater than 6-Log10 reduction was achieved within a 100 minutes exposure time. This pilot study puts forward a potential methodology for testing antimicrobial agents against biofilms and supports the efficacy of HPV.

Rapid in situ assessment of Cu-ion mediated effects and antibacterial efficacy of copper surfaces.

Release of metal ions from metal-based surfaces has been considered one of the main drivers of their antimicrobial activity. Here we describe a method that enables parallel assessment of metal ion release from solid metallic surfaces and antimicrobial efficacy of these surfaces in a short time period. The protocol involves placement of a small volume of bioluminescent bacteria onto the tested surface and direct measurement of bioluminescence at various time points. In this study, two recombinant Escherichia coli strains, one expressing bioluminescence constitutively and applicable for general antimicrobial testing, and the other induced by Cu ions, were selected. Decrease in bioluminescence of constitutive E. coli on the surfaces showed a good correlation with the decrease in bacterial viability. Response of Cu-inducible E. coli showed a correlation with Cu content in the tested surfaces but not with Cu dissolution suggesting the role of direct bacteria-surface contact in Cu ion-driven antibacterial effects. In summary, the presented protocol enables the analysis of microbial toxicity and bioavailability of surface-released metal ions directly on solid surfaces within 30-60 min. Although optimized for copper and copper alloy surfaces and E. coli, the method can be extended to other types of metallic surfaces and bacterial strains.

Viable-but-Nonculturable Listeria monocytogenes and Salmonella enterica Serovar Thompson Induced by Chlorine Stress Remain Infectious.

The microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-tagged Listeria monocytogenes and Salmonella enterica serovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viable L. monocytogenes and Salmonella Thompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed by using Caenorhabditis elegans Ingestion of VBNC pathogens by C. elegans resulted in a significant life span reduction (P = 0.0064 and P < 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturable L. monocytogenes treatments was observed. L. monocytogenes was visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected.IMPORTANCE Many bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogens Listeria monocytogenes and Salmonella enterica It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed in Caenorhabditis elegans that ingested these VBNC pathogens, with VBNC L. monocytogenes as infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.

Specialized cleaning associated with antimicrobial coatings for reduction of hospital-acquired infection: opinion of the COST Action Network AMiCI (CA15114).

Recognized issues with poor hand hygiene compliance among healthcare workers and reports of recontamination of previously chemically disinfected surfaces through hand contact emphasize the need for novel hygiene methods in addition to those currently available. One such approach involves antimicrobial (nano) coatings (AMCs), whereby integrated active ingredients are responsible for elimination of micro-organisms that come into contact with treated surfaces. While widely studied under laboratory conditions with promising results, studies under real-life healthcare conditions are scarce. The views of 75 contributors from 30 European countries were collated regarding specialized cleaning associated with AMCs for reduction of healthcare-associated infection. There was unanimous agreement that generation of scientific guidelines for cleaning of AMCs, using traditional or new processes, is needed. Specific topics included: understanding mechanisms of action of cleaning materials and their physical interactions with conventional coatings and AMCs; that assessments mimic the life cycle of coatings to determine the impact of repetitive cleaning and other aspects of ageing (e.g. exposure to sunlight); determining concentrations of AMC-derived biocides in effluents; and development of effective de-activation and sterilization treatments for cleaning effluents. Further, the consensus opinion was that, prior to widespread implementation of AMCs, there is a need for clarification of the varying responsibilities of involved clinical, healthcare management, cleaning services and environmental safety stakeholders.

Lack of Involvement of Fenton Chemistry in Death of Methicillin-Resistant and Methicillin-Sensitive Strains of Staphylococcus aureus and Destruction of Their Genomes on Wet or Dry Copper Alloy Surfaces.

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.

Human Coronavirus 229E Remains Infectious on Common Touch Surface Materials.

UNLABELLED: 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. IMPORTANCE: Respiratory viruses are responsible for more deaths globally than any other infectious agent. Animal coronaviruses that "host jump" to humans result in severe infections with high mortality, such as severe acute respiratory syndrome (SARS) and, more recently, Middle East respiratory syndrome (MERS). We show here that a closely related human coronavirus, 229E, which causes upper respiratory tract infection in healthy individuals and serious disease in patients with comorbidities, remained infectious on surface materials common to public and domestic areas for several days. The low infectious dose means that this is a significant infection risk to anyone touching a contaminated surface. However, rapid inactivation, irreversible destruction of viral RNA, and massive structural damage were observed in coronavirus exposed to copper and copper alloy surfaces. Incorporation of copper alloy surfaces in conjunction with effective cleaning regimens and good clinical practice could help to control transmission of respiratory coronaviruses, including MERS and SARS.