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Antimicrobial properties of a novel copper-based composite coating with potential for use in healthcare facilities

Antimicrobial properties of a novel copper-based composite coating with potential for use in... Background: Healthcare-associated infections (HAIs) have a major impact on public health worldwide. Particularly, hospital surfaces contaminated with bacterial pathogens are often the origin of both sporadic cases and outbreaks of HAIs. It has been demonstrated that copper surfaces reduce the microbial burden of high touch surfaces in the hospital environment. Here we report the antimicrobial characterization of a novel composite coating with embedded copper particles, named Copper Armour™. Methods: The Copper Armour™ bactericidal activity was evaluated in in vitro assays against several bacterial pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli O157:H7 and Listeria monocytogenes. Additionally, its antimicrobial properties were also evaluated in a pilot study over a nine-week period at an adult intensive care unit. For this, four high touch surfaces, including bed rails, overbed table, bedside table and IV Pole, were coated with Cooper Armour™, and its microbial burden was determined over a nine-week period. Results: Copper Armour™ coated samples showed an in vitro reduction in bacterial burden of > 99.9% compared to control samples. Moreover, pilot study results indicate that Copper Armour™ significantly reduces the level of microbial contamination on high-touch surfaces in the hospital environment, as compared with standard surfaces. Conclusions: Based on its antimicrobial properties, Copper Armour™ is a novel self-sanitizing coating that exhibits bactericidal activity against important human pathogens and significantly reduces the microbial burden of hospital surfaces. This composite could be used as a self-sanitizing coating to complement infection control strategies in healthcare facilities. Keywords: Antimicrobial copper, Copper-based composite, Self-sanitizing coating, High-touch surfaces, Healthcare- associated infections Background Multiple factors contribute to the incidence of HAIs, Healthcare-associated infections (HAIs) are the most including intrinsic patient conditions (e.g. their individ- frequent adverse event threatening the life of hospital- ual pathologies) and risk factors associated with the hos- ized patients worldwide [1]. HAIs have a major impact pital environment. Specifically, medical devices and on public health, as they increase the average length of hospital surfaces contaminated with pathogenic microor- hospital stays, morbidity and mortality [2, 3], and cause ganisms are often the origin of both sporadic cases and a significant increase in healthcare costs [4, 5]. outbreaks of HAIs [2, 6, 7]. Pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), * Correspondence: marisol@atacamalab.cl; claudio@atacamalab.cl; vancomycin-resistant Enterococcus spp. (VRE) and Clos- rvidal@uchile.cl tridium difficile, are able to colonize hospital surfaces, ATACAMALAB, Lampa, Chile and both spores and the vegetative form can persist on Programa de Microbiología y Micología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile these surfaces for months [7]. Therefore, hand hygiene Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 2 of 10 and routine and terminal cleaning of surfaces in contact Many types of microorganisms can persist for ex- with the patients are useful strategies to limit tended periods of time on high-touch surfaces; therefore, intra-hospital propagation of infectious agents [8, 9]. At this type of surfaces represent high risk spots for patho- present, the microbiological standard used to evaluate gen transmission and HAIs. In this context, a main con- and monitor terminal cleaning of hospital surfaces is a cern is to eliminate as many pathogenic microorganisms count of 250–500 aerobic colony-forming units (cfu) per as possible from these surfaces and limiting their trans- 100 cm [10, 11]. However, while deep cleaning may fer to patients [26] . Due to their antimicrobial proper- remove the majority of microorganisms present on hos- ties, metals, including copper, have been a focus of pital surfaces, they are susceptible to recontamination, interest as coating materials for surfaces. In this sce- which in some cases occurs in a very short period of nario, the aim of this study was to determine in situ time [12]. whether a composite based on copper significantly re- In 2008, the United States Environmental Protection duced the microbial load on coated surfaces in an adult Agency (US EPA) recognized copper as the first anti- intensive care unit (ICU) when compared to control (i.e. microbial metal. In in vitro assays, solid copper surfaces non-coated) surfaces and, in parallel, to determine if a killed 99.9% of microorganisms within two hours of con- composite based on copper has in vitro antimicrobial tact [13]. The rate of this antimicrobial activity has a activity against relevant pathogenic bacteria. magnitude of 7 to 8 logarithms per hour and generally no microorganisms are recovered after longer incubation Methods periods [14]. Likewise, copper particles exhibit potent Bacterial strains and culture conditions antimicrobial activity [15]. The bactericidal activity of The microorganisms used in this study were obtained copper is mainly attributed to the release of ions, which from American Type Culture Collection (ATCC) and affect the integrity of the membrane and/or the bacterial they include: Staphylococcus aureus (ATCC 29213), wall, generate intracellular oxidative stress and are geno- Pseudomonas aeruginosa (ATCC 27853), Escherichia coli toxic, resulting in the death of microorganisms [14, 15]. O157:H7 (ATCC 43895) and Listeria monocytogenes One advantage of copper as a bactericidal agent is the (ATCC 13932). S. aureus, P. aeruginosa and E. coli were low level of resistance among clinically relevant microor- routinely cultured in Trypticase Soy Broth (TSB, BD ganisms. Copper-resistant mechanisms are primarily Difco™, USA) and Trypticase Soy Agar (TSA, BD Difco™, found in environmental microorganisms living in USA) for 24–36 h at 37 ± 0.5 °C. L. monocytogenes was copper-rich niches, such as marine sediments and mines routinely cultured in Brain Heart Infusion Broth (BHI, [15, 16]. BD Difco™, USA) and BHI Agar (BD Difco™, USA) for Consequently, the number of studies evaluating the 24–48 h at 37 ± 0.5 °C. use of copper as a strategy for reducing the microbial burden in hospital environments and to prevent HAIs Formulation of copper Armour™ has increased in the past few years [11, 12, 17–23]. Re- Copper Armour™ is a composite material that is embed- sults from these studies indicate that hospital surfaces ded with copper particles in a methyl methacrylate resin coated with solid copper show sustained reduction in (matrix) evenly distributed in the matrix, so that copper microbial burden compared to control surfaces. Never- particles are always partially exposed on the surface. To theless, additional studies are necessary to determine the achieve this effect, at least four types of copper particles impact of using copper-coated surfaces on the incidence are used; as these particles differ in shape, apparent of HAIs. While some studies concluded that using densities (with a range of < 1–8g/cm ; Fig. 1a, b) and copper-coated surfaces reduces the rate of these infec- capacity to be compacted among themselves, when tions [11, 22], in others this reduction was not statisti- mixed together in a polymeric matrix they can be dis- cally significant [21]. Furthermore, heterogeneity in tributed homogeneously in the entire thickness of the study design and data analysis among existing studies composite structure. makes it hard to compare their results, and therefore to Three components were separately formulated. The draw definitive conclusions [24, 25]. Based on these ob- first component, a polymeric base, includes an agglom- servations, the use of copper-coated surfaces and med- erative or polymeric matrix with a dispersion of copper ical devices is a promising strategy for controlling HAIs. nanoparticles and microparticles < 20 μm; this dispersion This report summarizes the development and anti- is achieved by conventional methods using a high-shear microbial characterization of a composite material that mixing blade (Cowles), where nanoparticles < 0.1 μm includes copper particles, named Copper Armour™. Due were previously homogenized by an ultrasonic agitator. to its initial liquid state, this novel composite can be This semi-manufactured product is filtered using a used to impregnate various surfaces; after it dries (~ 2.5 200-mesh (74 μm) sieve prior to packaging. The second h) it provides a solid coating of 0.5–3.0 mm thick. component, the active component, is made from larger Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 3 of 10 A B CD Fig. 1 Formulation of Copper Armour™. a Copper Armour™ can be applied in liquid state on various substrates. At 25 °C, a 1 mm thick coating requires 2.5 h to dry. b Schematic composition of Copper Armour™. Shapes and sizes of Cu particles embedded in the methacrylate resin (matrix) are shown; the matrix acts as a liquid medium, providing adherence to the substrate and cohesion among components. Larger spherical Cu particles precipitate before curing of the matrix. Dendritic Cu particles act as a charge-conducting network. Smaller flakes Cu particles, float on the surface and become oriented in parallel, increasing the contact surface, thus, favoring the release of Cu ions. A bacterium is shown with its membrane degraded as a consequence of the antimicrobial activity of Cu. c Superficial topography of Copper Armour™. SEM analysis showed a homogenous distribution of copper particles in the matrix. d Chemical composition of Copper Armour™. EDAX analysis shows that Cu, carbon (C) and oxygen (O) are the main elements of the composite copper particles, up to 60 μm, that are dry packaged elements present by Energy Dispersive X-ray spectros- after sieving and drying to avoid agglomeration. The copy (EDAX). Samples were coated with gold (Au) to third component, a hardener, is separately packaged in a render them conductive. third container, depending on the selected agglomerate. The application of Copper Armour™ is performed In vitro evaluation of antimicrobial activity using a disperser, preferably electric from 200 to 600 The in vitro evaluation of antimicrobial activity was con- rpm, homogenizing the polymeric base with the active ducted based on two EPA protocols [27, 28], with slight component. Then, the hardener is added and homoge- modifications. The EPA designed these protocols to de- nized for at least one minute. This mixture must be ap- termine the efficacy of copper as a disinfectant, and to plied within 10 min following preparation, as after 15 quantify the continuous reduction of bacterial contamin- min it will begin to solidify. ation of non-porous surfaces containing copper and its For the assays described in this work, the Copper alloys. Armour™ formulation correspond to a 60/40 copper/ag- glomerate total weight ratio. The agglomerative methyl Test method of sanitizer activity (protocol 1) Two methacrylate resin used was DEGADUR 527 (Evonik batches of test samples (each one consisting of five 2 × 2 A.G., Germany), with powdered solid peroxide hardener. cm aluminum sheets coated with Copper Armour™) and Copper Armour™ formulations are protected by Patent ten control samples (2 × 2 cm aluminum sheets) were Cooperation Treaty international application number: evaluated per microorganism. Test and control samples PCT/CL2015/050058. were cleaned using 70% ethanol and washed using sterile distilled water. Each sample was placed in a Petri dish Electron microscopy and allowed to dry in a biological safety cabinet (Class II The superficial topography of Copper Armour™ was ana- type A2, NuAire, USA), followed by exposure to ultra- lyzed by scanning electron microscopy (SEM) using a violet light for 15 min per side. Hitachi SU 3500 microscope coupled to a series 410-M Bacterial culture media were supplemented with 5% detector, which allowed us to qualitatively analyze the heat-inactivated fetal calf serum (GIBCO, USA) and Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 4 of 10 0.01% Triton X-100 as organic sediment load. Initial in- using 1 ml of TPL solution, 0.1 ml of this solution was 7 8 ocula (10 to 10 cfu) were determined by serial dilutions plated on TSA and the absence of bacterial growth was in 1X phosphate-buffered saline (PBS) and plated in du- confirmed. Finally, each microorganism was inoculated plicate on TSA for 24–48 h at 37 ± 0.5 °C. Test samples in 1 ml of TPL solution, and it was determined that this and controls were inoculated with 0.02 ml of bacterial solution did not inhibit bacterial growth. culture spread over ~ 0.3 cm and allowed to dry for 20– 40 min. After 60 min of exposure (at room temperature) Test method of continuous reduction of bacterial to the challenging microorganisms, samples were trans- contamination (protocol 2) Two batches of test sam- ferred to 20 ml of neutralizing solution [TPL; Trypticase ples (each one consisting of three 2 × 2 cm aluminum Soy Broth plus Polysorbate 80 (1.5% v/v) and Lecithin sheets coated with Copper Armour™) and six control (0.07% v/v)], sonicated in an ultrasonic bath (Neytech samples (2 × 2 cm aluminum sheets) were evaluated per ultrasonic cleaner, Model 19H, USA) for 5 min and microorganism in a similar fashion to that described in turned to mix. Within 1 h, serial dilutions were per- Protocol 1. Samples were consecutively inoculated eight formed in PBS and plated in duplicate on TSA. After in- times, adding the challenging microorganism at 0, 3, 6, cubation for 24–48 h at 37 ± 0.5 °C, the number of cfu 9, 12, 15, 18 and 21 h. The antimicrobial efficacy was was counted. The number of cfu recovered per sample evaluated at 2, 6, 12, 18 and 24 h, corresponding to 1, 2, was determined taking into consideration the dilution 4, 6 and 8 inoculations. After exposure to bacteria, 20 ml (20x), using the following equation: cfu/sample = (A x D of TPL solution was added and samples were subjected xV) / V , where A = average cfu per sample, counted in to sonication in an ultrasonic bath and turned to mix. duplicate; D = dilution factor; V = volume of TPL solu- The determination of the number of cfu recovered per tion added; and V = volume plated. The percentage re- sample and the percent reduction was performed as de- duction in the number of cfu for test samples as scribed for protocol 1. Additionally, we performed the compared with the control samples was determined same sterility controls as previously described. using the following equation: % reduction = [(a-b) / a] × 100 where, a = geometric median of the number of cfu recovered in control samples; and b = geometric median Pilot study at an adult intensive care unit of the number of cfu recovered in the test samples. The study was conducted in two patient rooms (side by In addition, the following sterility control was per- side) within the adult ICU at the Hospital Clínico formed: 0.1 ml aliquots of culture media, PBS and TPL Universidad de Chile located in Santiago, Chile. One of solution were plated on TSA and the absence of bacter- the rooms was defined as the control and in the other ial growth was confirmed. One test and one control room, considered the intervention room, surfaces were sample, sterilized as previously described, were washed coated with Copper Armour™ (Fig. 2a). The following Fig. 2 Distribution of coated and sampled surfaces within the adult intensive care unit rooms. a Distribution of the sampled objects within the room. In the intervention room, the coated surfaces are shown in gold. b Copper Armour™ coated objects. (1) Bed rails, (2) Overbed table, (3) Bedside table and (4) IV Pole. Black arrows indicate where surface sampled were taken for each object Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 5 of 10 surfaces were coated with Copper Armour™: bed rails, Statistical analysis overbed table, bedside table and IV Pole (Fig. 2b). Upon Data on microbial burden obtained from hospital sur- admission, patients were randomly assigned to either the faces were analyzed for normality using the control or intervention (Copper Armour™) room. Hand Shapiro-Wilk test. As the data did not follow a normal hygiene and cleaning protocols remained unaltered dur- distribution, the non-parametric Mann-Whitney U test ing the study. (one-tailed) was used to determine if the microbial bur- The sampling protocol was performed over a den of Copper Armour™ coated surfaces was signifi- nine-week period, during which the first week (basal cantly lower compared to control surfaces. Additionally, week) was dedicated to methodology adjustments. Data differences in the frequency of microbial burden, re- obtained during this week were not included in statis- ported as > 250 cfu/100 cm surface, between control tical analysis and are not shown. Rooms were sampled and Copper Armour™ coated surfaces was analyzed on the same day and at the same time (before morning using the Fisher’s exact test or the Pearson χ test (if all cleaning) every week throughout the study. Surfaces expected frequencies were ≥ 5). A P-value of < 0.05 was were sampled in duplicate (Fig. 2b, black arrows) using considered statistically significant; statistical analysis was sterile plastic templates of 2 × 12.5 cm, in the case of bed performed in GraphPad Prism version 6.00 (GraphPad rails the IV Pole, or 5 × 5 cm, in the case of the overbed Software, La Jolla California USA). and bedside tables. PBS humidified sterile dressing was vigorously scrubbed 10 times horizontally and 10 times Results vertically, covering the whole sampling area (25 cm ). Characterization of the microstructure and chemical Each dressing was placed in a 50 ml sterile polypropyl- composition of copper Armour™ ene centrifuge tube. Within 2 h, three ml of PBS/LT SEM analysis of samples coated with Copper Armour™ (0.5% Tween 80 and 0.07% lecithin) were added to each showed a homogenous distribution of copper particles in centrifuge tube, vortexed for 1 min, and allowed to settle the methacrylate matrix (Fig. 1c). Additionally, qualitative for 5 min. Subsequently, 100 μl aliquots were plated on chemical analysis indicated that the main component in 5% sheep blood agar to estimate the total aerobic micro- the coating was copper (Cu), while carbon (C) and oxygen bial burden present on sampled surface; mannitol salt (O) were the main matrix components (Fig. 1d). agar (BD Difco™, USA) to estimate the number of cfu of Staphylococcus spp.; MacConkey agar (BD Difco™, USA) In vitro evaluation of the antimicrobial properties of to determine the number of cfu of Gram-negative bacilli; copper Armour™ chromogenic agar (BBLTM-BD CHROMagar MRSA™, Challenging microorganisms for the evaluation of Becton Dickinson, USA) to estimate the number of cfu in vitro bactericidal activity were Staphylococcus aureus, of MRSA; bile esculin agar (Becton Dickinson, USA) Pseudomonas aeruginosa, Escherichia coli O157:H7 and supplemented with vancomycin (6 μg/ml) to determine Listeria monocytogenes. All experiments conducted with the number of cfu of VRE and Sabouraud agar (Becton Copper Armour™ coated samples showed a reduction, Dickinson, USA) supplemented with chloramphenicol after 1 h of contact, in bacterial burden of > 99.9% com- (CAF) to estimate the cfu of yeast / fungi. Plates were pared to control samples (Table 1). Additionally, we de- incubated for 24–48 h at 37 ± 0.5 °C and the number of termined that after consecutive inoculations over 24 h, cfu were determined. The number of cfu recovered per Copper Armour™ coated samples continued to reduce sample was reported as cfu/100 cm . the microbial burden by > 99.9% compared to control Table 1 Reduction in bacterial burden after 1 h of contact with Copper Armour™ as compared to control surfaces Microorganism Batch Inoculum Number of cfu recovered per sample * Reduction (cfu) (%) ** Control Copper ArmourTm 7 6 6 6 6 6 S. aureus 1 4.3 × 10 1.2 × 10 ; 3.0 × 10 ; 1.8 × 10 ; 1.9 × 10 ; 3.1 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 6 6 6 6 6 2 1.5 × 10 1.4 × 10 ; 1.1 × 10 ; 1.1 × 10 ; 2.0 × 10 ; 1.2 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 8 7 7 6 7 6 P. aeruginosa 1 1.6 × 10 4.4 × 10 ; 2.1 × 10 ; 7.2 × 10 ; 4.4 × 10 ; 9.3 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 8 7 7 7 7 7 2 1.8 × 10 1.1 × 10 ; 2.8 × 10 ; 1.2 × 10 ; 1.0 × 10 ; 1.1 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 5 6 6 6 5 E. coli O157:H7 1 1.9 × 10 8.1 × 10 ; 4.3 × 10 ; 4.1 × 10 ; 5.4 × 10 ; 9.6 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 6 6 6 6 5 2 2.4 × 10 5.3 × 10 ; 3.8 × 10 ; 2.4 × 10 ; 2.5 × 10 ; 7.9 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 6 6 6 6 6 L. monocytogenes 1 3.2 × 10 7.2 × 10 ; 8.7 × 10 ; 9.4 × 10 ; 7.3 × 10 ; 6.3 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 6 6 6 6 6 2 1.6 × 10 9.7 × 10 ; 8.0 × 10 ; 7.3 × 10 ; 7.7 × 10 ; 7.8 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 * Each value corresponds to the average of duplicates of cfu recovered in each one of the five samples evaluated per production batch. ** As compared with control samples Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 6 of 10 samples (Table 2). Thus, Copper Armour™ continuously (56%; p = 0.045). Additionally, the average number of reduced contamination caused by the bacteria evaluated cfu/100 cm for Staphylococcus spp. was lower on here. Copper Armour™ coated surfaces compared to control surfaces (Table3); however, this reduction was only Evaluation of the antimicrobial properties of copper statistically significant in the case of bed rails (88.9%; p Armour™ at an adult intensive care unit < 0.001). It is important to mention that during the Copper Armour™ coated surfaces (Fig. 2) showed a re- study S. aureus was not recovered from any surface, and duction of the aerobic microbial burden compared to only one Copper Armour™ coated surface was positive control surfaces; this reduction was statistically signifi- for Gram negative bacilli (720 cfu/100 cm ) and 2 for cant for bed rails (66%; p = 0.018) and the overbed Table VRE (both samples with 120 cfu/100 cm ); due to these Table 2 Continuous reduction of bacterial burden over 24 h of contact with Copper Armour™ as compared to control surfaces Microorganism Time Batch Number of cfu recovered per sample * Reduction (h) (%) ** Controls Copper ArmourTm 7 7 5 5 5 S. aureus Inoculum: 2.0 × 10 –5.0 × 10 21 3.8 × 10 ; 3.1 × 10 ; 3.9 × 10 < 1;< 1;< 1 > 99.9 5 5 5 2 4.0 × 10 ; 4.1 × 10 ; 3.2 × 10 < 1;< 1;< 1 6 6 6 61 1.8 × 10 ; 1.8 × 10 ; 2.0 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 1.1 × 10 ; 1.5 × 10 ; 1.2 × 10 < 1;< 1;< 1 6 6 6 12 1 4.4 × 10 ; 4.5 × 10 ; 4.5 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 3.9 × 10 ; 4.4 × 10 ; 4.0 × 10 < 1;< 1;< 1 6 6 6 18 1 6.6 × 10 ; 5.9 × 10 ; 6.1 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 7.9 × 10 ; 6.4 × 10 ; 6.8 × 10 < 1;< 1;< 1 7 7 7 24 1 2.0 × 10 ; 1.0 × 10 ; 1.3 × 10 < 1;< 1;< 1 > 99.9 7 6 6 2 1.0 × 10 ; 9.4 × 10 ; 9.9 × 10 < 1;< 1;< 1 8 8 6 6 6 P. aeruginosa Inoculum: 1.6 × 10 –1.8 × 10 21 7.4 × 10 ; 7.4 × 10 ; 7.2 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 6.2 × 10 ; 6.8 × 10 ; 6.5 × 10 < 1;< 1;< 1 6 6 6 61 7.6 × 10 ; 7.8 × 10 ; 7.6 × 10 5800; < 1; 2000 > 99.9 6 6 6 2 8.2 × 10 ; 7.8 × 10 ; 7.9 × 10 < 1;< 1;< 1 7 7 7 12 1 1.6 × 10 ; 1.4 × 10 ; 1.3 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 1.0 × 10 ; 1.1 × 10 ; 2.0 × 10 < 1;< 1;< 1 7 7 7 18 1 4.8 × 10 ; 4.8 × 10 ; 4.3 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 5.0 × 10 ; 5.2 × 10 ; 4.9 × 10 < 1;< 1;< 1 8 8 7 24 1 1.1 × 10 ; 1.0 × 10 ; 9.6 × 10 < 1;< 1;< 1 > 99.9 8 8 8 2 1.3 × 10 ; 2.0 × 10 ; 1.9 × 10 < 1;< 1;< 1 7 7 5 5 5 E. coli O157:H7 Inoculum: 2.0 × 10 –4.0 × 10 21 2.8 × 10 ; 3.1 × 10 ; 3.0 × 10 < 1;< 1;< 1 > 99.9 5 5 5 2 3.5 × 10 ; 3.5 × 10 ; 3.3 × 10 < 1;< 1;< 1 6 6 6 61 1.6 × 10 ; 1.7 × 10 ; 1.6 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 2.2 × 10 ; 1.9 × 10 ; 2.0 × 10 < 1;< 1;< 1 6 6 6 12 1 4.6 × 10 ; 4.6 × 10 ; 4.5 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 4.2 × 10 ; 4.9 × 10 ; 4.3 × 10 < 1;< 1;< 1 6 7 6 18 1 9.8 × 10 ; 1.1 × 10 ; 9.5 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 1.2 × 10 ; 1.0 × 10 ; 1.0 × 10 < 1;< 1;< 1 7 7 7 24 1 3.2 × 10 ; 3.0 × 10 ; 2.9 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 3.9 × 10 ; 4.1 × 10 ; 4.0 × 10 < 1;< 1;< 1 7 7 7 7 7 L. monocytogenes Inoculum: 1.0 × 10 –5.0 × 10 21 1,6 × 10 ; 2,1 × 10 ; 2,2 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 1,5 × 10 ; 1,4 × 10 ; 2,1 × 10 < 1;< 1;< 1 7 7 7 61 3,0 × 10 ; 4,1 × 10 ; 4,3 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 3,6 × 10 ; 3,7 × 10 ; 2,7 × 10 < 1;< 1;< 1 7 7 7 12 1 4,8 × 10 ; 5,1 × 10 ; 6,0 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 4,7 × 10 ; 4,7 × 10 ; 4,1 × 10 < 1;< 1;< 1 7 7 7 18 1 9,8 × 10 ; 9,1 × 10 ; 9,8 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 9,2 × 10 ; 9,4 × 10 ; 9,0 × 10 < 1;< 1;< 1 8 8 8 24 1 2,2 × 10 ; 2,0 × 10 ; 2,1 × 10 < 1;< 1;< 1 > 99.9 8 8 8 2 1,9 × 10 ; 1,2 × 10 ; 1,2 × 10 < 1;< 1;< 1 * Each value corresponds to the average of duplicates of cfu recovered in each one of the three samples evaluated per production batch. ** As compared with control samples Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 7 of 10 low detection rates, these microorganisms were not in- Our results indicate that Copper Armour™ may be cluded in statistical analyses. In contrast, we did not ob- used as a self-sanitizing coating to modify existing hos- serve a reduction in the average burden of yeasts / fungi pital surfaces, avoiding the structural restrictions im- on Copper Armour™ coated surfaces as compared to posed by a change to solid copper. Due to its initial control surfaces. Nevertheless, the isolation of these mi- liquid state and subsequent hardening, this composite croorganisms was sporadic overall, with values < 250 can be used to coat surfaces of various sizes, shapes and cfu/100 cm during the study. compositions, which reduces the cost and quantity of In agreement with previous results, the frequency of sam- copper required. ples with a microbial burden > 250 cfu/100 cm was lower The in vitro evaluation of the antimicrobial properties in the case of Copper Armour™ coated surfaces compared of Copper Armour™ showed that this composite material to control surfaces (Fig. 3); this difference was statistically exhibits a potent bactericidal activity against S. aureus, significant for bed rails (40.6% Copper Armour™ versus P. aeruginosa, E. coli O157:H7 and L. monocytogenes.As 68.8% control; p = 0.023) and the overbed Table (35.7% reported for solid copper, Copper Armour™ killed more Copper Armour™ versus 75% control; p = 0.030) (Table 4). than 99.9% of these microorganisms after one hour of Furthermore, the overall frequency of control surfaces with contact, as well as after consecutive inoculations over a microbial burden of > 250 cfu/100 cm was significantly 24 h (Table 1 & Table 2). It is noteworthy that two of greater than Copper Armour coated surfaces, 60% (48/80) these microorganisms, S. aureus and P. aeruginosa, are versus 33.3% (p = 0.007). Thus, Copper Armour™ exhibits among the principal pathogens causing HAIs worldwide antimicrobial properties able to decrease the microbial bur- [34–36]. Moreover, the emergence of resistant and mul- den of high-touch surfaces in a hospital environment. tiresistant bacteria makes it necessary to develop new Therefore, compared to control surfaces, Copper Armour™ biocidal materials and agents able to limit the dissemin- coated surfaces were more likely to meet the threshold re- ation and, at the same time, contribute to the elimin- quired for successful terminal cleaning (i.e. < 250 cfu/100 ation of these pathogens. cm ), indicating that the use of this composite could con- We also evaluated the Copper Armour™ antimicro- tribute to schemes and practices aimed at controlling HAIs. bial properties in a hospital environment. Our pilot study indicated that Copper Armour™ reduces the mi- Discussion crobial burden of hospital surfaces, even under It has been demonstrated that high-touch surfaces in the present day protocols of extreme hygiene. A study by hospital environment are an important reservoir for in- Attaway et al. [6]showedthat bed railsinICUsare fectious agents causing HAIs [6, 29]. In this context, a rapidly colonized after cleaning with two commercial considerable number of studies have provided experi- disinfectants, exceeding the threshold of 250 cfu/100 mental evidence indicating that hospital surfaces coated cm after 2.5 h. In that study, the average microbial with copper have lower microbial burden levels com- burden found on bed rails before cleaning was 4.756 2 2 pared to standard surfaces, which in some cases have cfu/100 cm (median 1.665 cfu/100 cm ). Likewise, our been associated with a reduction in the incidence of results showed that control bed rails had an average HAIs [11, 12, 17–23]. However, while most of these microbial burden of 3.323 cfu/100 cm (median 1.440 studies have been conducted using solid copper and its cfu/100 cm )(Table 3). On the contrary, Copper alloys, the in vitro and in situ evaluation of polymeric Armour™ coated bed rails showed an average micro- matrices and composites containing copper particles has bial burden of 1.129 cfu/100 cm (median 120 cfu/100 been limited [30–33]. cm ), which corresponds to a significant reduction Fig. 3 Frequency distribution of microbial burden on Copper Armour™ coated surfaces and control surfaces. The microbial burden observed for 2 2 each sample was classified into three categories: below the detection threshold (green), 1 to 250 cfu/100 cm (yellow) or > 250 cfu/100 cm (red) Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 8 of 10 Table 3 Bacterial burden on Copper Armour™ coated surfaces and control surfaces during 8 weeks of pilot study in an adult intensive care unit Evaluated object Copper Armour™ Control P value % Reduction 2 2 2 2 n Average cfu/100 cm Media cfu/100 cm n Average cfu/100 cm Median cfu/100 cm Total aerobic microbial load Bed rails 32 1129 120 32 3323 1440 0.018 * 66.0 Overbed Table 14 762,9 0 16 1755 960 0,045 * 56.5 Bedside Table 16 1793 60 16 2108 120 0,303 14.9 IV Pole 16 157,5 0 16 337,5 120 0,195 53.5 Staphylococcus spp. Bed rails 32 270 0 32 2445 300 0,001 ** 88.9 Overbed Table 14 462,9 0 16 720 240 0,106 35.7 Bedside Table 16 270 0 16 997,5 0 0,289 72.9 IV Pole 16 22,5 0 16 60 0 0,231 62.5 Yeasts/Fungi Bed rails 32 697,5 0 32 195,0 0 –– Overbed Table 14 68,5 0 16 15,00 0 –– Bedside Table 16 630 0 16 1155 0 0,279 45.5 IV Pole 16 15 0 16 37,5 0 0,367 60 * p < 0.05, ** p < 0.001 established using Mann-Whitney U test (one-tailed) (66%; p = 0.018) compared to control bed rails. Thus, average burden of Staphylococcus spp. compared to con- Copper Armour™ exhibits antimicrobial properties trol bed rails (Table 3). able to decrease the microbial burden of high-touch The overbed table is another Copper Armour™ coated surfaces in a hospital environment. Therefore, com- surface in which a significant reduction (56%, p = 0.045) pared to control surfaces, Copper Armour™ coated of microbial burden was observed compared to the con- surfaces were more likely to meet the threshold re- trol overbed table. Besides, a lower average burden of quired for successful terminal cleaning (i.e. < 250 cfu/ Staphylococcus spp. was observed in the Copper 100 cm ), indicating that the use of this composite Armour™ coated overbed table compared to the control could contribute to schemes and practices aimed at overbed table, but in this case, the reduction, while controlling HAIs. showing a trend, was not significant (p = 0.105); this is It must be noted that two previous studies demon- likely due to the fact that the average burden of these strated that bed rails of solid copper showed a signifi- microorganism on the control surface was also low. Pre- cantly lower average microbial burden compared to vious studies have also shown that solid copper coated control bed rails [12, 37]. Also, in agreement with our overbed tables have lower level of microbial burden results, in those studies it was determined that Staphylo- compared to standard surfaces [37]. coccus spp. were the main bacterial group contaminating An intriguing result was the average microbial burden ICU bed rails. In fact, Copper Armour™ coated bed rails of the Copper Armour™ coated bedside tables compared showed a significant (88.9%, p < 0.001) reduction in the to the control. In this case, only a small and Table 4 Frequency of a microbial burden of > 250 cfu/100 cm on Copper Armour™ coated surfaces and control surfaces Evaluated Copper Armour™ Control P value objects 2 2 n Number (%) of samples having > 250 cfu/100 cm n Number (%) of samples having > 250 cfu/100 cm Bed rails 32 13 (40.6) 32 22 (68.8) 0.023 * Overbed Table 14 ** 5 (35.7) 16 12 (75) 0.030 * Bedside Table 16 6 (37.5) 16 7 (43.7) 0.718 IV Pole 16 2 (12.5) 16 7 (43.7) 0.113 Total 78 26 (33.3) 80 48 (60) 0.001 ** * p < 0.05, ** p < 0.001 established using either Pearson χ or Fisher’s Exact tests **Two samples were discarded because the surface was contaminated with blood Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 9 of 10 non-significant reduction (p = 0.289) of contamination Acknowledgements Not applicable. levels was observed (Table 3 & Table 4). A possible ex- planation for this result is that objects brought into the Funding hospital, which escape cleaning schemes, are constantly The authors gratefully acknowledge the financial support of Corporación de Fomento a la Producción (CORFO), Grant ID 17ITE2–82627. placed on the bedside table (Fig. 2b). It has been reported that among the objects located Availability of data and materials within a patient’s room, the IV pole shows, in general, Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. the lowest average microbial burden [18, 37]. This was also observed in the present study. It is likely Authors’ contribution that for this reason we were not able to observe MG, CR and RMV conceptualized and designed the study. CA and MP sampling and processing in the microbiological laboratory. RV performed the differences between the average microbial burden of a electron microscopy analyses. RG, MC and MB coordinated and design the Copper Armour™ coated IV pole and the control study in situ at the Hospital Clínico Universidad de Chile. RMV and DAM data surface. Nevertheless, 87.5% (14/16) of the samples acquisition, data analysis, data interpretation, revised the manuscript, prepared figures and tables. All authors contributed to the editing and from the Copper Armour™ coated IV Pole showed approved the final manuscript version. levels < 250 cfu/100 cm as compared to a 56.3% (9/ 16) of control samples (Fig. 3). This suggests that, in Ethics approval The Ethics Committee of the Hospital Clínico de la Universidad de Chile the case of surfaces exposed to low levels of contam- approved the study protocols and an informed consent was not required to ination, the main benefit provided by Copper obtain samples from hospital surfaces. Armour™ wouldbeto extendedprotectiontimeof Consent for publication the terminal cleaning. Not applicable. The pilot study also attempted to investigate the anti- fungal properties of Copper Armour™. Nevertheless, we Competing interests MG and CR are Directors of ATACAMALAB, a for profit Company aiming to were not able to complete this aim as isolation of fungi/ develop energy efficient products including novel uses for copper. yeast was sporadic and with low numbers of cfu/100 cm . Therefore, in order to evaluate this property, it Publisher’sNote would be necessary to implement a different methodo- Springer Nature remains neutral with regard to jurisdictional claims in logical design. published maps and institutional affiliations. Finally, our pilot study did not include parameters, Author details such as whether the room was occupied / unoccupied 1 Programa de Microbiología y Micología, Instituto de Ciencias Biomédicas, each day or epidemiological data of the patients. Future Facultad de Medicina, Universidad de Chile, Santiago, Chile. Instituto de Química, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, studies, that are longer in duration and that include dif- Valparaíso, Chile. Unidad de Cuidados Intensivos, Facultad de Medicina, ferent hospital surfaces beyond those tested here, and 4 Hospital Clínico Universidad de Chile, Santiago, Chile. ATACAMALAB, Lampa, that also consider patient factors are necessary to further Chile. Instituto Milenio de Inmunología e Inmunoterapia, Facultad de Medicina, Universidad de Chile, Santiago, Chile. evaluate the possible impact of Copper Armour™ on the incidence of HAIs. Received: 14 August 2018 Accepted: 17 December 2018 Conclusions References 1. Allegranzi B, Nejad SB, Combescure C, Graafmans W, Attar H, Donaldson L, Our study suggests that Copper Amour TM, a novel et al. Burden of endemic health-care-associated infection in developing self-sanitizing coating, exhibits bactericidal activity countries: systematic review and meta-analysis. 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Atlanta: centers for cfu: colony-forming units; EDAX: Energy Dispersive X-ray spectroscopy; disease control and Prevention; 2009. Available from: http://www.cdc.gov/ GIBCO: Grand Island Biological Company; HAI: Healthcare-associated hai/pdfs/hai/scott_costpaper.pdf infection; ICU: Intensive Care Unit; MRSA: Methicillin-Resistant Staphylococcus 5. Pola B, Nercelles P, Pohlenza M, Otaiza F. Costo de las infecciones aureus; PBS: Phosphate-Buffered Saline; PBS/LT: Phosphate-Buffered Saline/ intrahospitalarias en hospitales chilenos de alta y mediana complejidad. Rev Tween and Lecithin; PCT: Patent Cooperation Treaty; SEM: Scanning Electron Chil infectología. Sociedad Chilena de Infectología. 2003;20:285–90. Microscopy; TM: Trade Mark; TPL: Trypticase Soy Broth plus Polysorbate and 6. Attaway HH, Fairey S, Steed LL, Salgado CD, Michels HT, Schmidt MG. Lecithin; TSA: Trypticase Soy Agar; TSB: Trypticase Soy Broth; US EPA: United Intrinsic bacterial burden associated with intensive care unit hospital beds: States Environmental Protection Agency; VRE: Vancomycin-Resistant effects of disinfection on population recovery and mitigation of potential Enterococcus spp infection risk. Am J Infect Control. Elsevier Inc. 2012;40:907–12. Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 10 of 10 7. Kramer A, Schwebke I, Kampf G. How long do nosocomial pathogens 30. Palza H, Quijada R, Delgado K. Antimicrobial polymer composites with persist on inanimate surfaces? A systematic review. BMC Infect Dis. 2006;6: copper micro- and nanoparticles: effect of particle size and polymer matrix. 1–8. J Bioact Compat Polym. 2015;30:366–80. 8. Pittet D, Donaldson L. Clean care is safer care: the first global challenge of the 31. Thomas SF, Rooks P, Rudin F, Atkinson S, Goddard P, Bransgrove R, et al. 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A modified ATP benchmark for agent. 2011;50–54. evaluating the cleaning of some hospital environmental surfaces. J Hosp 34. Álvarez F, Palomar M, Insausti J, Olaechea P, Cerdá E, De MS, et al. Infecciones Infect. 2008;69:156–63. nosocomiales por Staphylococcus aureus en pacientes críticos en unidades de 11. Salgado CD, Sepkowitz KA, John JF, Cantey JR, Attaway HH, Freeman KD, cuidados intensivos. Med Clin (Barc) Elsevier. 2006;126:641–6. et al. Copper surfaces reduce the rate of healthcare-acquired infections in 35. Dantes R, Mu Y, Belflower R, Aragon D, Dumyati G, Harrison LH, et al. the intensive care unit. Infect Control Hosp Epidemiol. 2013;34:479–86. National Burden of Invasive Methicillin-Resistant 2013;30333. 12. Schmidt MG, Attaway HH, Fairey SE, Steed LL, Michels HT, Salgado CD. 36. Nathwani D, Raman G, Sulham K, Gavaghan M, Menon V. 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J Hosp Infect Elsevier Ltd. 2009;73:378–85. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antimicrobial Resistance & Infection Control Springer Journals

Antimicrobial properties of a novel copper-based composite coating with potential for use in healthcare facilities

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Springer Journals
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Copyright © 2019 by The Author(s).
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Biomedicine; Medical Microbiology; Drug Resistance; Infectious Diseases
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2047-2994
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10.1186/s13756-018-0456-4
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Abstract

Background: Healthcare-associated infections (HAIs) have a major impact on public health worldwide. Particularly, hospital surfaces contaminated with bacterial pathogens are often the origin of both sporadic cases and outbreaks of HAIs. It has been demonstrated that copper surfaces reduce the microbial burden of high touch surfaces in the hospital environment. Here we report the antimicrobial characterization of a novel composite coating with embedded copper particles, named Copper Armour™. Methods: The Copper Armour™ bactericidal activity was evaluated in in vitro assays against several bacterial pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli O157:H7 and Listeria monocytogenes. Additionally, its antimicrobial properties were also evaluated in a pilot study over a nine-week period at an adult intensive care unit. For this, four high touch surfaces, including bed rails, overbed table, bedside table and IV Pole, were coated with Cooper Armour™, and its microbial burden was determined over a nine-week period. Results: Copper Armour™ coated samples showed an in vitro reduction in bacterial burden of > 99.9% compared to control samples. Moreover, pilot study results indicate that Copper Armour™ significantly reduces the level of microbial contamination on high-touch surfaces in the hospital environment, as compared with standard surfaces. Conclusions: Based on its antimicrobial properties, Copper Armour™ is a novel self-sanitizing coating that exhibits bactericidal activity against important human pathogens and significantly reduces the microbial burden of hospital surfaces. This composite could be used as a self-sanitizing coating to complement infection control strategies in healthcare facilities. Keywords: Antimicrobial copper, Copper-based composite, Self-sanitizing coating, High-touch surfaces, Healthcare- associated infections Background Multiple factors contribute to the incidence of HAIs, Healthcare-associated infections (HAIs) are the most including intrinsic patient conditions (e.g. their individ- frequent adverse event threatening the life of hospital- ual pathologies) and risk factors associated with the hos- ized patients worldwide [1]. HAIs have a major impact pital environment. Specifically, medical devices and on public health, as they increase the average length of hospital surfaces contaminated with pathogenic microor- hospital stays, morbidity and mortality [2, 3], and cause ganisms are often the origin of both sporadic cases and a significant increase in healthcare costs [4, 5]. outbreaks of HAIs [2, 6, 7]. Pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), * Correspondence: marisol@atacamalab.cl; claudio@atacamalab.cl; vancomycin-resistant Enterococcus spp. (VRE) and Clos- rvidal@uchile.cl tridium difficile, are able to colonize hospital surfaces, ATACAMALAB, Lampa, Chile and both spores and the vegetative form can persist on Programa de Microbiología y Micología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile these surfaces for months [7]. Therefore, hand hygiene Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 2 of 10 and routine and terminal cleaning of surfaces in contact Many types of microorganisms can persist for ex- with the patients are useful strategies to limit tended periods of time on high-touch surfaces; therefore, intra-hospital propagation of infectious agents [8, 9]. At this type of surfaces represent high risk spots for patho- present, the microbiological standard used to evaluate gen transmission and HAIs. In this context, a main con- and monitor terminal cleaning of hospital surfaces is a cern is to eliminate as many pathogenic microorganisms count of 250–500 aerobic colony-forming units (cfu) per as possible from these surfaces and limiting their trans- 100 cm [10, 11]. However, while deep cleaning may fer to patients [26] . Due to their antimicrobial proper- remove the majority of microorganisms present on hos- ties, metals, including copper, have been a focus of pital surfaces, they are susceptible to recontamination, interest as coating materials for surfaces. In this sce- which in some cases occurs in a very short period of nario, the aim of this study was to determine in situ time [12]. whether a composite based on copper significantly re- In 2008, the United States Environmental Protection duced the microbial load on coated surfaces in an adult Agency (US EPA) recognized copper as the first anti- intensive care unit (ICU) when compared to control (i.e. microbial metal. In in vitro assays, solid copper surfaces non-coated) surfaces and, in parallel, to determine if a killed 99.9% of microorganisms within two hours of con- composite based on copper has in vitro antimicrobial tact [13]. The rate of this antimicrobial activity has a activity against relevant pathogenic bacteria. magnitude of 7 to 8 logarithms per hour and generally no microorganisms are recovered after longer incubation Methods periods [14]. Likewise, copper particles exhibit potent Bacterial strains and culture conditions antimicrobial activity [15]. The bactericidal activity of The microorganisms used in this study were obtained copper is mainly attributed to the release of ions, which from American Type Culture Collection (ATCC) and affect the integrity of the membrane and/or the bacterial they include: Staphylococcus aureus (ATCC 29213), wall, generate intracellular oxidative stress and are geno- Pseudomonas aeruginosa (ATCC 27853), Escherichia coli toxic, resulting in the death of microorganisms [14, 15]. O157:H7 (ATCC 43895) and Listeria monocytogenes One advantage of copper as a bactericidal agent is the (ATCC 13932). S. aureus, P. aeruginosa and E. coli were low level of resistance among clinically relevant microor- routinely cultured in Trypticase Soy Broth (TSB, BD ganisms. Copper-resistant mechanisms are primarily Difco™, USA) and Trypticase Soy Agar (TSA, BD Difco™, found in environmental microorganisms living in USA) for 24–36 h at 37 ± 0.5 °C. L. monocytogenes was copper-rich niches, such as marine sediments and mines routinely cultured in Brain Heart Infusion Broth (BHI, [15, 16]. BD Difco™, USA) and BHI Agar (BD Difco™, USA) for Consequently, the number of studies evaluating the 24–48 h at 37 ± 0.5 °C. use of copper as a strategy for reducing the microbial burden in hospital environments and to prevent HAIs Formulation of copper Armour™ has increased in the past few years [11, 12, 17–23]. Re- Copper Armour™ is a composite material that is embed- sults from these studies indicate that hospital surfaces ded with copper particles in a methyl methacrylate resin coated with solid copper show sustained reduction in (matrix) evenly distributed in the matrix, so that copper microbial burden compared to control surfaces. Never- particles are always partially exposed on the surface. To theless, additional studies are necessary to determine the achieve this effect, at least four types of copper particles impact of using copper-coated surfaces on the incidence are used; as these particles differ in shape, apparent of HAIs. While some studies concluded that using densities (with a range of < 1–8g/cm ; Fig. 1a, b) and copper-coated surfaces reduces the rate of these infec- capacity to be compacted among themselves, when tions [11, 22], in others this reduction was not statisti- mixed together in a polymeric matrix they can be dis- cally significant [21]. Furthermore, heterogeneity in tributed homogeneously in the entire thickness of the study design and data analysis among existing studies composite structure. makes it hard to compare their results, and therefore to Three components were separately formulated. The draw definitive conclusions [24, 25]. Based on these ob- first component, a polymeric base, includes an agglom- servations, the use of copper-coated surfaces and med- erative or polymeric matrix with a dispersion of copper ical devices is a promising strategy for controlling HAIs. nanoparticles and microparticles < 20 μm; this dispersion This report summarizes the development and anti- is achieved by conventional methods using a high-shear microbial characterization of a composite material that mixing blade (Cowles), where nanoparticles < 0.1 μm includes copper particles, named Copper Armour™. Due were previously homogenized by an ultrasonic agitator. to its initial liquid state, this novel composite can be This semi-manufactured product is filtered using a used to impregnate various surfaces; after it dries (~ 2.5 200-mesh (74 μm) sieve prior to packaging. The second h) it provides a solid coating of 0.5–3.0 mm thick. component, the active component, is made from larger Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 3 of 10 A B CD Fig. 1 Formulation of Copper Armour™. a Copper Armour™ can be applied in liquid state on various substrates. At 25 °C, a 1 mm thick coating requires 2.5 h to dry. b Schematic composition of Copper Armour™. Shapes and sizes of Cu particles embedded in the methacrylate resin (matrix) are shown; the matrix acts as a liquid medium, providing adherence to the substrate and cohesion among components. Larger spherical Cu particles precipitate before curing of the matrix. Dendritic Cu particles act as a charge-conducting network. Smaller flakes Cu particles, float on the surface and become oriented in parallel, increasing the contact surface, thus, favoring the release of Cu ions. A bacterium is shown with its membrane degraded as a consequence of the antimicrobial activity of Cu. c Superficial topography of Copper Armour™. SEM analysis showed a homogenous distribution of copper particles in the matrix. d Chemical composition of Copper Armour™. EDAX analysis shows that Cu, carbon (C) and oxygen (O) are the main elements of the composite copper particles, up to 60 μm, that are dry packaged elements present by Energy Dispersive X-ray spectros- after sieving and drying to avoid agglomeration. The copy (EDAX). Samples were coated with gold (Au) to third component, a hardener, is separately packaged in a render them conductive. third container, depending on the selected agglomerate. The application of Copper Armour™ is performed In vitro evaluation of antimicrobial activity using a disperser, preferably electric from 200 to 600 The in vitro evaluation of antimicrobial activity was con- rpm, homogenizing the polymeric base with the active ducted based on two EPA protocols [27, 28], with slight component. Then, the hardener is added and homoge- modifications. The EPA designed these protocols to de- nized for at least one minute. This mixture must be ap- termine the efficacy of copper as a disinfectant, and to plied within 10 min following preparation, as after 15 quantify the continuous reduction of bacterial contamin- min it will begin to solidify. ation of non-porous surfaces containing copper and its For the assays described in this work, the Copper alloys. Armour™ formulation correspond to a 60/40 copper/ag- glomerate total weight ratio. The agglomerative methyl Test method of sanitizer activity (protocol 1) Two methacrylate resin used was DEGADUR 527 (Evonik batches of test samples (each one consisting of five 2 × 2 A.G., Germany), with powdered solid peroxide hardener. cm aluminum sheets coated with Copper Armour™) and Copper Armour™ formulations are protected by Patent ten control samples (2 × 2 cm aluminum sheets) were Cooperation Treaty international application number: evaluated per microorganism. Test and control samples PCT/CL2015/050058. were cleaned using 70% ethanol and washed using sterile distilled water. Each sample was placed in a Petri dish Electron microscopy and allowed to dry in a biological safety cabinet (Class II The superficial topography of Copper Armour™ was ana- type A2, NuAire, USA), followed by exposure to ultra- lyzed by scanning electron microscopy (SEM) using a violet light for 15 min per side. Hitachi SU 3500 microscope coupled to a series 410-M Bacterial culture media were supplemented with 5% detector, which allowed us to qualitatively analyze the heat-inactivated fetal calf serum (GIBCO, USA) and Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 4 of 10 0.01% Triton X-100 as organic sediment load. Initial in- using 1 ml of TPL solution, 0.1 ml of this solution was 7 8 ocula (10 to 10 cfu) were determined by serial dilutions plated on TSA and the absence of bacterial growth was in 1X phosphate-buffered saline (PBS) and plated in du- confirmed. Finally, each microorganism was inoculated plicate on TSA for 24–48 h at 37 ± 0.5 °C. Test samples in 1 ml of TPL solution, and it was determined that this and controls were inoculated with 0.02 ml of bacterial solution did not inhibit bacterial growth. culture spread over ~ 0.3 cm and allowed to dry for 20– 40 min. After 60 min of exposure (at room temperature) Test method of continuous reduction of bacterial to the challenging microorganisms, samples were trans- contamination (protocol 2) Two batches of test sam- ferred to 20 ml of neutralizing solution [TPL; Trypticase ples (each one consisting of three 2 × 2 cm aluminum Soy Broth plus Polysorbate 80 (1.5% v/v) and Lecithin sheets coated with Copper Armour™) and six control (0.07% v/v)], sonicated in an ultrasonic bath (Neytech samples (2 × 2 cm aluminum sheets) were evaluated per ultrasonic cleaner, Model 19H, USA) for 5 min and microorganism in a similar fashion to that described in turned to mix. Within 1 h, serial dilutions were per- Protocol 1. Samples were consecutively inoculated eight formed in PBS and plated in duplicate on TSA. After in- times, adding the challenging microorganism at 0, 3, 6, cubation for 24–48 h at 37 ± 0.5 °C, the number of cfu 9, 12, 15, 18 and 21 h. The antimicrobial efficacy was was counted. The number of cfu recovered per sample evaluated at 2, 6, 12, 18 and 24 h, corresponding to 1, 2, was determined taking into consideration the dilution 4, 6 and 8 inoculations. After exposure to bacteria, 20 ml (20x), using the following equation: cfu/sample = (A x D of TPL solution was added and samples were subjected xV) / V , where A = average cfu per sample, counted in to sonication in an ultrasonic bath and turned to mix. duplicate; D = dilution factor; V = volume of TPL solu- The determination of the number of cfu recovered per tion added; and V = volume plated. The percentage re- sample and the percent reduction was performed as de- duction in the number of cfu for test samples as scribed for protocol 1. Additionally, we performed the compared with the control samples was determined same sterility controls as previously described. using the following equation: % reduction = [(a-b) / a] × 100 where, a = geometric median of the number of cfu recovered in control samples; and b = geometric median Pilot study at an adult intensive care unit of the number of cfu recovered in the test samples. The study was conducted in two patient rooms (side by In addition, the following sterility control was per- side) within the adult ICU at the Hospital Clínico formed: 0.1 ml aliquots of culture media, PBS and TPL Universidad de Chile located in Santiago, Chile. One of solution were plated on TSA and the absence of bacter- the rooms was defined as the control and in the other ial growth was confirmed. One test and one control room, considered the intervention room, surfaces were sample, sterilized as previously described, were washed coated with Copper Armour™ (Fig. 2a). The following Fig. 2 Distribution of coated and sampled surfaces within the adult intensive care unit rooms. a Distribution of the sampled objects within the room. In the intervention room, the coated surfaces are shown in gold. b Copper Armour™ coated objects. (1) Bed rails, (2) Overbed table, (3) Bedside table and (4) IV Pole. Black arrows indicate where surface sampled were taken for each object Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 5 of 10 surfaces were coated with Copper Armour™: bed rails, Statistical analysis overbed table, bedside table and IV Pole (Fig. 2b). Upon Data on microbial burden obtained from hospital sur- admission, patients were randomly assigned to either the faces were analyzed for normality using the control or intervention (Copper Armour™) room. Hand Shapiro-Wilk test. As the data did not follow a normal hygiene and cleaning protocols remained unaltered dur- distribution, the non-parametric Mann-Whitney U test ing the study. (one-tailed) was used to determine if the microbial bur- The sampling protocol was performed over a den of Copper Armour™ coated surfaces was signifi- nine-week period, during which the first week (basal cantly lower compared to control surfaces. Additionally, week) was dedicated to methodology adjustments. Data differences in the frequency of microbial burden, re- obtained during this week were not included in statis- ported as > 250 cfu/100 cm surface, between control tical analysis and are not shown. Rooms were sampled and Copper Armour™ coated surfaces was analyzed on the same day and at the same time (before morning using the Fisher’s exact test or the Pearson χ test (if all cleaning) every week throughout the study. Surfaces expected frequencies were ≥ 5). A P-value of < 0.05 was were sampled in duplicate (Fig. 2b, black arrows) using considered statistically significant; statistical analysis was sterile plastic templates of 2 × 12.5 cm, in the case of bed performed in GraphPad Prism version 6.00 (GraphPad rails the IV Pole, or 5 × 5 cm, in the case of the overbed Software, La Jolla California USA). and bedside tables. PBS humidified sterile dressing was vigorously scrubbed 10 times horizontally and 10 times Results vertically, covering the whole sampling area (25 cm ). Characterization of the microstructure and chemical Each dressing was placed in a 50 ml sterile polypropyl- composition of copper Armour™ ene centrifuge tube. Within 2 h, three ml of PBS/LT SEM analysis of samples coated with Copper Armour™ (0.5% Tween 80 and 0.07% lecithin) were added to each showed a homogenous distribution of copper particles in centrifuge tube, vortexed for 1 min, and allowed to settle the methacrylate matrix (Fig. 1c). Additionally, qualitative for 5 min. Subsequently, 100 μl aliquots were plated on chemical analysis indicated that the main component in 5% sheep blood agar to estimate the total aerobic micro- the coating was copper (Cu), while carbon (C) and oxygen bial burden present on sampled surface; mannitol salt (O) were the main matrix components (Fig. 1d). agar (BD Difco™, USA) to estimate the number of cfu of Staphylococcus spp.; MacConkey agar (BD Difco™, USA) In vitro evaluation of the antimicrobial properties of to determine the number of cfu of Gram-negative bacilli; copper Armour™ chromogenic agar (BBLTM-BD CHROMagar MRSA™, Challenging microorganisms for the evaluation of Becton Dickinson, USA) to estimate the number of cfu in vitro bactericidal activity were Staphylococcus aureus, of MRSA; bile esculin agar (Becton Dickinson, USA) Pseudomonas aeruginosa, Escherichia coli O157:H7 and supplemented with vancomycin (6 μg/ml) to determine Listeria monocytogenes. All experiments conducted with the number of cfu of VRE and Sabouraud agar (Becton Copper Armour™ coated samples showed a reduction, Dickinson, USA) supplemented with chloramphenicol after 1 h of contact, in bacterial burden of > 99.9% com- (CAF) to estimate the cfu of yeast / fungi. Plates were pared to control samples (Table 1). Additionally, we de- incubated for 24–48 h at 37 ± 0.5 °C and the number of termined that after consecutive inoculations over 24 h, cfu were determined. The number of cfu recovered per Copper Armour™ coated samples continued to reduce sample was reported as cfu/100 cm . the microbial burden by > 99.9% compared to control Table 1 Reduction in bacterial burden after 1 h of contact with Copper Armour™ as compared to control surfaces Microorganism Batch Inoculum Number of cfu recovered per sample * Reduction (cfu) (%) ** Control Copper ArmourTm 7 6 6 6 6 6 S. aureus 1 4.3 × 10 1.2 × 10 ; 3.0 × 10 ; 1.8 × 10 ; 1.9 × 10 ; 3.1 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 6 6 6 6 6 2 1.5 × 10 1.4 × 10 ; 1.1 × 10 ; 1.1 × 10 ; 2.0 × 10 ; 1.2 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 8 7 7 6 7 6 P. aeruginosa 1 1.6 × 10 4.4 × 10 ; 2.1 × 10 ; 7.2 × 10 ; 4.4 × 10 ; 9.3 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 8 7 7 7 7 7 2 1.8 × 10 1.1 × 10 ; 2.8 × 10 ; 1.2 × 10 ; 1.0 × 10 ; 1.1 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 5 6 6 6 5 E. coli O157:H7 1 1.9 × 10 8.1 × 10 ; 4.3 × 10 ; 4.1 × 10 ; 5.4 × 10 ; 9.6 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 6 6 6 6 5 2 2.4 × 10 5.3 × 10 ; 3.8 × 10 ; 2.4 × 10 ; 2.5 × 10 ; 7.9 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 6 6 6 6 6 L. monocytogenes 1 3.2 × 10 7.2 × 10 ; 8.7 × 10 ; 9.4 × 10 ; 7.3 × 10 ; 6.3 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 7 6 6 6 6 6 2 1.6 × 10 9.7 × 10 ; 8.0 × 10 ; 7.3 × 10 ; 7.7 × 10 ; 7.8 × 10 < 1;< 1;< 1;< 1;< 1 > 99.9 * Each value corresponds to the average of duplicates of cfu recovered in each one of the five samples evaluated per production batch. ** As compared with control samples Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 6 of 10 samples (Table 2). Thus, Copper Armour™ continuously (56%; p = 0.045). Additionally, the average number of reduced contamination caused by the bacteria evaluated cfu/100 cm for Staphylococcus spp. was lower on here. Copper Armour™ coated surfaces compared to control surfaces (Table3); however, this reduction was only Evaluation of the antimicrobial properties of copper statistically significant in the case of bed rails (88.9%; p Armour™ at an adult intensive care unit < 0.001). It is important to mention that during the Copper Armour™ coated surfaces (Fig. 2) showed a re- study S. aureus was not recovered from any surface, and duction of the aerobic microbial burden compared to only one Copper Armour™ coated surface was positive control surfaces; this reduction was statistically signifi- for Gram negative bacilli (720 cfu/100 cm ) and 2 for cant for bed rails (66%; p = 0.018) and the overbed Table VRE (both samples with 120 cfu/100 cm ); due to these Table 2 Continuous reduction of bacterial burden over 24 h of contact with Copper Armour™ as compared to control surfaces Microorganism Time Batch Number of cfu recovered per sample * Reduction (h) (%) ** Controls Copper ArmourTm 7 7 5 5 5 S. aureus Inoculum: 2.0 × 10 –5.0 × 10 21 3.8 × 10 ; 3.1 × 10 ; 3.9 × 10 < 1;< 1;< 1 > 99.9 5 5 5 2 4.0 × 10 ; 4.1 × 10 ; 3.2 × 10 < 1;< 1;< 1 6 6 6 61 1.8 × 10 ; 1.8 × 10 ; 2.0 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 1.1 × 10 ; 1.5 × 10 ; 1.2 × 10 < 1;< 1;< 1 6 6 6 12 1 4.4 × 10 ; 4.5 × 10 ; 4.5 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 3.9 × 10 ; 4.4 × 10 ; 4.0 × 10 < 1;< 1;< 1 6 6 6 18 1 6.6 × 10 ; 5.9 × 10 ; 6.1 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 7.9 × 10 ; 6.4 × 10 ; 6.8 × 10 < 1;< 1;< 1 7 7 7 24 1 2.0 × 10 ; 1.0 × 10 ; 1.3 × 10 < 1;< 1;< 1 > 99.9 7 6 6 2 1.0 × 10 ; 9.4 × 10 ; 9.9 × 10 < 1;< 1;< 1 8 8 6 6 6 P. aeruginosa Inoculum: 1.6 × 10 –1.8 × 10 21 7.4 × 10 ; 7.4 × 10 ; 7.2 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 6.2 × 10 ; 6.8 × 10 ; 6.5 × 10 < 1;< 1;< 1 6 6 6 61 7.6 × 10 ; 7.8 × 10 ; 7.6 × 10 5800; < 1; 2000 > 99.9 6 6 6 2 8.2 × 10 ; 7.8 × 10 ; 7.9 × 10 < 1;< 1;< 1 7 7 7 12 1 1.6 × 10 ; 1.4 × 10 ; 1.3 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 1.0 × 10 ; 1.1 × 10 ; 2.0 × 10 < 1;< 1;< 1 7 7 7 18 1 4.8 × 10 ; 4.8 × 10 ; 4.3 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 5.0 × 10 ; 5.2 × 10 ; 4.9 × 10 < 1;< 1;< 1 8 8 7 24 1 1.1 × 10 ; 1.0 × 10 ; 9.6 × 10 < 1;< 1;< 1 > 99.9 8 8 8 2 1.3 × 10 ; 2.0 × 10 ; 1.9 × 10 < 1;< 1;< 1 7 7 5 5 5 E. coli O157:H7 Inoculum: 2.0 × 10 –4.0 × 10 21 2.8 × 10 ; 3.1 × 10 ; 3.0 × 10 < 1;< 1;< 1 > 99.9 5 5 5 2 3.5 × 10 ; 3.5 × 10 ; 3.3 × 10 < 1;< 1;< 1 6 6 6 61 1.6 × 10 ; 1.7 × 10 ; 1.6 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 2.2 × 10 ; 1.9 × 10 ; 2.0 × 10 < 1;< 1;< 1 6 6 6 12 1 4.6 × 10 ; 4.6 × 10 ; 4.5 × 10 < 1;< 1;< 1 > 99.9 6 6 6 2 4.2 × 10 ; 4.9 × 10 ; 4.3 × 10 < 1;< 1;< 1 6 7 6 18 1 9.8 × 10 ; 1.1 × 10 ; 9.5 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 1.2 × 10 ; 1.0 × 10 ; 1.0 × 10 < 1;< 1;< 1 7 7 7 24 1 3.2 × 10 ; 3.0 × 10 ; 2.9 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 3.9 × 10 ; 4.1 × 10 ; 4.0 × 10 < 1;< 1;< 1 7 7 7 7 7 L. monocytogenes Inoculum: 1.0 × 10 –5.0 × 10 21 1,6 × 10 ; 2,1 × 10 ; 2,2 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 1,5 × 10 ; 1,4 × 10 ; 2,1 × 10 < 1;< 1;< 1 7 7 7 61 3,0 × 10 ; 4,1 × 10 ; 4,3 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 3,6 × 10 ; 3,7 × 10 ; 2,7 × 10 < 1;< 1;< 1 7 7 7 12 1 4,8 × 10 ; 5,1 × 10 ; 6,0 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 4,7 × 10 ; 4,7 × 10 ; 4,1 × 10 < 1;< 1;< 1 7 7 7 18 1 9,8 × 10 ; 9,1 × 10 ; 9,8 × 10 < 1;< 1;< 1 > 99.9 7 7 7 2 9,2 × 10 ; 9,4 × 10 ; 9,0 × 10 < 1;< 1;< 1 8 8 8 24 1 2,2 × 10 ; 2,0 × 10 ; 2,1 × 10 < 1;< 1;< 1 > 99.9 8 8 8 2 1,9 × 10 ; 1,2 × 10 ; 1,2 × 10 < 1;< 1;< 1 * Each value corresponds to the average of duplicates of cfu recovered in each one of the three samples evaluated per production batch. ** As compared with control samples Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 7 of 10 low detection rates, these microorganisms were not in- Our results indicate that Copper Armour™ may be cluded in statistical analyses. In contrast, we did not ob- used as a self-sanitizing coating to modify existing hos- serve a reduction in the average burden of yeasts / fungi pital surfaces, avoiding the structural restrictions im- on Copper Armour™ coated surfaces as compared to posed by a change to solid copper. Due to its initial control surfaces. Nevertheless, the isolation of these mi- liquid state and subsequent hardening, this composite croorganisms was sporadic overall, with values < 250 can be used to coat surfaces of various sizes, shapes and cfu/100 cm during the study. compositions, which reduces the cost and quantity of In agreement with previous results, the frequency of sam- copper required. ples with a microbial burden > 250 cfu/100 cm was lower The in vitro evaluation of the antimicrobial properties in the case of Copper Armour™ coated surfaces compared of Copper Armour™ showed that this composite material to control surfaces (Fig. 3); this difference was statistically exhibits a potent bactericidal activity against S. aureus, significant for bed rails (40.6% Copper Armour™ versus P. aeruginosa, E. coli O157:H7 and L. monocytogenes.As 68.8% control; p = 0.023) and the overbed Table (35.7% reported for solid copper, Copper Armour™ killed more Copper Armour™ versus 75% control; p = 0.030) (Table 4). than 99.9% of these microorganisms after one hour of Furthermore, the overall frequency of control surfaces with contact, as well as after consecutive inoculations over a microbial burden of > 250 cfu/100 cm was significantly 24 h (Table 1 & Table 2). It is noteworthy that two of greater than Copper Armour coated surfaces, 60% (48/80) these microorganisms, S. aureus and P. aeruginosa, are versus 33.3% (p = 0.007). Thus, Copper Armour™ exhibits among the principal pathogens causing HAIs worldwide antimicrobial properties able to decrease the microbial bur- [34–36]. Moreover, the emergence of resistant and mul- den of high-touch surfaces in a hospital environment. tiresistant bacteria makes it necessary to develop new Therefore, compared to control surfaces, Copper Armour™ biocidal materials and agents able to limit the dissemin- coated surfaces were more likely to meet the threshold re- ation and, at the same time, contribute to the elimin- quired for successful terminal cleaning (i.e. < 250 cfu/100 ation of these pathogens. cm ), indicating that the use of this composite could con- We also evaluated the Copper Armour™ antimicro- tribute to schemes and practices aimed at controlling HAIs. bial properties in a hospital environment. Our pilot study indicated that Copper Armour™ reduces the mi- Discussion crobial burden of hospital surfaces, even under It has been demonstrated that high-touch surfaces in the present day protocols of extreme hygiene. A study by hospital environment are an important reservoir for in- Attaway et al. [6]showedthat bed railsinICUsare fectious agents causing HAIs [6, 29]. In this context, a rapidly colonized after cleaning with two commercial considerable number of studies have provided experi- disinfectants, exceeding the threshold of 250 cfu/100 mental evidence indicating that hospital surfaces coated cm after 2.5 h. In that study, the average microbial with copper have lower microbial burden levels com- burden found on bed rails before cleaning was 4.756 2 2 pared to standard surfaces, which in some cases have cfu/100 cm (median 1.665 cfu/100 cm ). Likewise, our been associated with a reduction in the incidence of results showed that control bed rails had an average HAIs [11, 12, 17–23]. However, while most of these microbial burden of 3.323 cfu/100 cm (median 1.440 studies have been conducted using solid copper and its cfu/100 cm )(Table 3). On the contrary, Copper alloys, the in vitro and in situ evaluation of polymeric Armour™ coated bed rails showed an average micro- matrices and composites containing copper particles has bial burden of 1.129 cfu/100 cm (median 120 cfu/100 been limited [30–33]. cm ), which corresponds to a significant reduction Fig. 3 Frequency distribution of microbial burden on Copper Armour™ coated surfaces and control surfaces. The microbial burden observed for 2 2 each sample was classified into three categories: below the detection threshold (green), 1 to 250 cfu/100 cm (yellow) or > 250 cfu/100 cm (red) Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 8 of 10 Table 3 Bacterial burden on Copper Armour™ coated surfaces and control surfaces during 8 weeks of pilot study in an adult intensive care unit Evaluated object Copper Armour™ Control P value % Reduction 2 2 2 2 n Average cfu/100 cm Media cfu/100 cm n Average cfu/100 cm Median cfu/100 cm Total aerobic microbial load Bed rails 32 1129 120 32 3323 1440 0.018 * 66.0 Overbed Table 14 762,9 0 16 1755 960 0,045 * 56.5 Bedside Table 16 1793 60 16 2108 120 0,303 14.9 IV Pole 16 157,5 0 16 337,5 120 0,195 53.5 Staphylococcus spp. Bed rails 32 270 0 32 2445 300 0,001 ** 88.9 Overbed Table 14 462,9 0 16 720 240 0,106 35.7 Bedside Table 16 270 0 16 997,5 0 0,289 72.9 IV Pole 16 22,5 0 16 60 0 0,231 62.5 Yeasts/Fungi Bed rails 32 697,5 0 32 195,0 0 –– Overbed Table 14 68,5 0 16 15,00 0 –– Bedside Table 16 630 0 16 1155 0 0,279 45.5 IV Pole 16 15 0 16 37,5 0 0,367 60 * p < 0.05, ** p < 0.001 established using Mann-Whitney U test (one-tailed) (66%; p = 0.018) compared to control bed rails. Thus, average burden of Staphylococcus spp. compared to con- Copper Armour™ exhibits antimicrobial properties trol bed rails (Table 3). able to decrease the microbial burden of high-touch The overbed table is another Copper Armour™ coated surfaces in a hospital environment. Therefore, com- surface in which a significant reduction (56%, p = 0.045) pared to control surfaces, Copper Armour™ coated of microbial burden was observed compared to the con- surfaces were more likely to meet the threshold re- trol overbed table. Besides, a lower average burden of quired for successful terminal cleaning (i.e. < 250 cfu/ Staphylococcus spp. was observed in the Copper 100 cm ), indicating that the use of this composite Armour™ coated overbed table compared to the control could contribute to schemes and practices aimed at overbed table, but in this case, the reduction, while controlling HAIs. showing a trend, was not significant (p = 0.105); this is It must be noted that two previous studies demon- likely due to the fact that the average burden of these strated that bed rails of solid copper showed a signifi- microorganism on the control surface was also low. Pre- cantly lower average microbial burden compared to vious studies have also shown that solid copper coated control bed rails [12, 37]. Also, in agreement with our overbed tables have lower level of microbial burden results, in those studies it was determined that Staphylo- compared to standard surfaces [37]. coccus spp. were the main bacterial group contaminating An intriguing result was the average microbial burden ICU bed rails. In fact, Copper Armour™ coated bed rails of the Copper Armour™ coated bedside tables compared showed a significant (88.9%, p < 0.001) reduction in the to the control. In this case, only a small and Table 4 Frequency of a microbial burden of > 250 cfu/100 cm on Copper Armour™ coated surfaces and control surfaces Evaluated Copper Armour™ Control P value objects 2 2 n Number (%) of samples having > 250 cfu/100 cm n Number (%) of samples having > 250 cfu/100 cm Bed rails 32 13 (40.6) 32 22 (68.8) 0.023 * Overbed Table 14 ** 5 (35.7) 16 12 (75) 0.030 * Bedside Table 16 6 (37.5) 16 7 (43.7) 0.718 IV Pole 16 2 (12.5) 16 7 (43.7) 0.113 Total 78 26 (33.3) 80 48 (60) 0.001 ** * p < 0.05, ** p < 0.001 established using either Pearson χ or Fisher’s Exact tests **Two samples were discarded because the surface was contaminated with blood Montero et al. Antimicrobial Resistance and Infection Control (2019) 8:3 Page 9 of 10 non-significant reduction (p = 0.289) of contamination Acknowledgements Not applicable. levels was observed (Table 3 & Table 4). A possible ex- planation for this result is that objects brought into the Funding hospital, which escape cleaning schemes, are constantly The authors gratefully acknowledge the financial support of Corporación de Fomento a la Producción (CORFO), Grant ID 17ITE2–82627. placed on the bedside table (Fig. 2b). It has been reported that among the objects located Availability of data and materials within a patient’s room, the IV pole shows, in general, Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. the lowest average microbial burden [18, 37]. This was also observed in the present study. It is likely Authors’ contribution that for this reason we were not able to observe MG, CR and RMV conceptualized and designed the study. CA and MP sampling and processing in the microbiological laboratory. RV performed the differences between the average microbial burden of a electron microscopy analyses. RG, MC and MB coordinated and design the Copper Armour™ coated IV pole and the control study in situ at the Hospital Clínico Universidad de Chile. RMV and DAM data surface. Nevertheless, 87.5% (14/16) of the samples acquisition, data analysis, data interpretation, revised the manuscript, prepared figures and tables. All authors contributed to the editing and from the Copper Armour™ coated IV Pole showed approved the final manuscript version. levels < 250 cfu/100 cm as compared to a 56.3% (9/ 16) of control samples (Fig. 3). This suggests that, in Ethics approval The Ethics Committee of the Hospital Clínico de la Universidad de Chile the case of surfaces exposed to low levels of contam- approved the study protocols and an informed consent was not required to ination, the main benefit provided by Copper obtain samples from hospital surfaces. Armour™ wouldbeto extendedprotectiontimeof Consent for publication the terminal cleaning. Not applicable. The pilot study also attempted to investigate the anti- fungal properties of Copper Armour™. Nevertheless, we Competing interests MG and CR are Directors of ATACAMALAB, a for profit Company aiming to were not able to complete this aim as isolation of fungi/ develop energy efficient products including novel uses for copper. yeast was sporadic and with low numbers of cfu/100 cm . Therefore, in order to evaluate this property, it Publisher’sNote would be necessary to implement a different methodo- Springer Nature remains neutral with regard to jurisdictional claims in logical design. published maps and institutional affiliations. Finally, our pilot study did not include parameters, Author details such as whether the room was occupied / unoccupied 1 Programa de Microbiología y Micología, Instituto de Ciencias Biomédicas, each day or epidemiological data of the patients. Future Facultad de Medicina, Universidad de Chile, Santiago, Chile. Instituto de Química, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, studies, that are longer in duration and that include dif- Valparaíso, Chile. Unidad de Cuidados Intensivos, Facultad de Medicina, ferent hospital surfaces beyond those tested here, and 4 Hospital Clínico Universidad de Chile, Santiago, Chile. ATACAMALAB, Lampa, that also consider patient factors are necessary to further Chile. Instituto Milenio de Inmunología e Inmunoterapia, Facultad de Medicina, Universidad de Chile, Santiago, Chile. evaluate the possible impact of Copper Armour™ on the incidence of HAIs. Received: 14 August 2018 Accepted: 17 December 2018 Conclusions References 1. Allegranzi B, Nejad SB, Combescure C, Graafmans W, Attar H, Donaldson L, Our study suggests that Copper Amour TM, a novel et al. Burden of endemic health-care-associated infection in developing self-sanitizing coating, exhibits bactericidal activity countries: systematic review and meta-analysis. 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Antimicrobial Resistance & Infection Control – Springer Journals

Published: Jan 5, 2019

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