Antimicrobial Activity of Copper Alloys Against Invasive Multidrug-Resistant Nosocomial Pathogens

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1 Curr Microbiol (215) 71: DOI 1.17/s Antimicrobial Activity of Copper Alloys Against Invasive Multidrug-Resistant Nosocomial Pathogens Ozgen Koseoglu Eser 1 Alper Ergin 2 Gulsen Hascelik 1 Received: 8 December 214 / Accepted: 18 April 215 / Published online: 5 June 215 Springer Science+Business Media New York 215 Abstract The emergence and spread of antibiotic resistance demanded novel approaches for the prevention of nosocomial infections, and metallic copper surfaces have been suggested as an alternative for the control of multidrug-resistant (MDR) bacteria in surfaces in the hospital environment. This study aimed to evaluate the antimicrobial activity of copper material for invasive MDR nosocomial pathogens isolated over time, in comparison to stainless steel. Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) (n:4), OXA-23 and OXA- 58 positive, MDR Acinetobacter baumannii (n:6) and Pseudomonas aeruginosa (n:4) were evaluated. The antimicrobial activity of coupons containing 99 % copper and a brass alloy containing 63 % copper was assessed against stainless steel. All the materials demonstrated statistically significant differences within each other for the logarithmic reduction of microorganisms. Among the three materials, the highest reduction of microorganisms was seen in 99 % copper and the least in stainless steel. The result was statistically significant especially for, 2, and 4 h (P =.5). 99 % copper showed a bactericidal effect at less than 1 h for MRSA and at 2 h for P. aeruginosa. 63 % copper showed a bactericidal effect at 24 h for P. aeruginosa strains only. Stainless steel surfaces exhibited a bacteriostatic effect after 6 h for P. aeruginosa strains only. 99 % copper reduced the number of bacteria used significantly, produced a bactericidal effect and was more effective than & Ozgen Koseoglu Eser ozgen.eser@hacettepe.edu.tr 1 2 Department of Medical Microbiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey School of Health Services, Hacettepe University, Ankara, Turkey 63 % copper. The use of metallic copper material could aid in reducing the concentration of bacteria, especially for invasive nosocomial pathogens on hard surfaces in the hospital environment. Introduction Multidrug-resistant (MDR) and extremely drug-resistant (XDR) pathogens are a serious and emerging threat to patients in healthcare settings and have a significant impact on mortality, cost and length of hospital stay [6, 11, 19]. Most frequent MDR pathogens are described as ESCAPE pathogens by taking the first initials of Enterococcus, Staphylococcus aureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae [19]. Overuse of antibiotics due to increasing levels of antimicrobial resistance especially against these pathogens continues to reduce the number of the few effective antimicrobial agents. The hospital environment is an important source for contributing to cross-infection. Effective removal of nosocomial pathogens from the hospital environment can be reduced by disinfectants and detergents. Stainless steel is widely used in the healthcare environment because it is corrosion resistant to most cleaning materials and always appears clean although it has no antimicrobial activity. The progressive erosion of therapeutic actions has been forced to receive renewed attention against biocidal surfaces in reducing nosocomial infections. The use of copper alloy biocidal surfaces could help to prevent the spread of microorganisms in healthcare settings [24]. Copper is an essential element at low concentrations as a cofactor for living organisms. The utilization of copper alloys at high concentrations proved to exhibit toxic effects by contact

2 292 O. Koseoglu Eser et al.: Antimicrobial Activity of Copper Alloys Against Invasive killing most microorganisms [5, 18, 22]. Copper and many copper alloys have already been registered at the U.S. Environmental Protection Agency (EPA) in 28 as the first solid antimicrobial material. A number of hospital trials in which rooms have been fitted with copper alloy door handles, bathroom fixtures, and tabletops have been conducted or are ongoing [12]. The aim of the present study was to evaluate the antimicrobial activity of copper surfaces for invasive MDR nosocomial pathogens isolated over time in comparison to stainless steel. Materials and Methods Bacterial Strains Invasive clinical isolates of multidrug-resistant (MDR), OXA-23 (n:5) and OXA-58 (n:1) positive A. baumannii (n:6), P. aeruginosa (n:4) and methicillin-resistant Staphylococcus aureus (MRSA)(n:4) isolated from blood cultures were evaluated. Species identification was performed using the BD Phoenix system (Sparks, MD, USA). The Cefoxitin disk screen test was used for the confirmation of MRSA strains as suggested by CLSI [3]. Antibiotic susceptibility of the isolates was determined by the microdilution method in accordance with guidelines of CLSI [3]. All the tested A. baumannii and P. aeruginosa isolates were resistant to all groups of antibiotics (beta-lactam and beta-lactamase combinations including third generation cephalosporins and carbapenems, as well as fluoroquinolones) except colistin. The MRSA isolates were also resistant to clindamycin, erythromycin, trimethoprim-sulphamethoxazole, and ciprofloxacin. Polymerase chain reaction with specific primers was used for confirmation of the presence of bla OXA-23 and bla OXA-58 genes. Standard strains of three microorganisms S. aureus ATCC 25923, A. baumannii DSM 37, and P. aeruginosa ATCC were also used in all experiments. Copper and Stainless Coupons The antimicrobial activity of copper, metal coupons (ACR Chemicals, Turkey) (1 cm 2 ) containing 99 % Cu (Cu99) and a brass alloy containing 63 % Cu (Cu63Zn37) was assessed. Coupons were cleaned before use by the modified method of Noyce et al. [16]. The coupons were degreased and cleaned by vortexing in 1 ml of acetone containing 3 glass beads of 2 mm diameter. After cleaning, the coupons were sterilized with absolute ethanol and flamed in a Bunsen burner. Sterilized coupons were placed in sterile glass tubes until use. Stainless steel coupons were sterilized using the same method and used as control in all experiments. Bacterial Viability Tests Organisms grown on sheep blood agar were adjusted to.5 McFarland standard. Ten microlitres of this suspension ( cfu) was inoculated on each of the three coupons, which were then incubated at room temperature (2 25 C) for, 2, 4, 6, and 24 h. Time was considered the time the aliquot dried. At each time interval coupons were aseptically removed and placed into 5 ml aliquots of sterile phosphate buffered saline (PBS) containing 2 glass beads and vortexed 6 s. The aliquots were serially diluted and plated onto sheep blood agar plates in duplicate and incubated at 37 C for 18 h for the determination of surviving bacteria. The lower limit of detection was.69 log 1 cfu/cm 2. The survival over time on each of the surfaces was expressed as mean log 1 cfu/cm 2 of all isolates of the same species at each time point. Bacteriostatic activity was defined as a C2 to\3 log 1 reduction and bactericidal activity as a C 3 log 1 reduction in the cfu from the inoculum at time on that surface. Statistical Analysis Effects of coupon, bacteria and coupon-bacteria relations in accordance with bacterial log reduction were compared using two-way variance analysis. Mean viable counts (log 1 cfu/cm 2 ) after incubation at each time interval with each coupon were compared statistically using one-way analysis of variance. The Bonferroni test was used to determine the reason of significant variance analysis differences within the materials. All differences with P \.5 were considered statistically significant. All statistical analyses were determined using the IBM SPSS 2. Statistics program. Results All the materials (Cu99, Cu63, and stainless steel) demonstrated statistically significant differences within each other for logarithmic reduction of microorganisms (P \.1). All three materials showed statistical significance for the log reduction of microorganisms (P \.1). Among the three materials, the highest reduction of microorganisms was seen in Cu99 (-3.17 ±.118) and the least in stainless steel (-.526 ±.118). At times, 2, and 4 h, only Cu99 showed a significant difference for log bacterial reduction (P \.5) and no

3 O. Koseoglu Eser et al.: Antimicrobial Activity of Copper Alloys Against Invasive 293 significance for Cu63 and stainless steel among all microorganisms. At 6 and 24 h, there was no significant difference between the three materials for all microorganisms (P [.5). The inactivation of microorganisms on different surfaces against time is shown in Fig. 1. Bactericidal and bacteriostatic effects of Cu63, Cu99, and SS against microorganisms in each time interval, meaning the difference between mean starting inoculum and mean viable cell count (D log 1 cfu/cm 2 ) of microorganisms, are shown in Table 1. Cu99 showed a bactericidal effect of less than 1 h for MRSA, 2 h for P. aeruginosa and exhibited a closer bactericidal range effect against A. baumannii starting from time zero (Table 1). Cu63 showed a bactericidal effect at 24 h for only P. aeruginosa among the isolates studied. Stainless steel surfaces exhibited a bacteriostatic effect after 6 h for P. aeruginosa strains only (Table 1). Discussion Environmental surfaces in the hospital setting act as an important reservoir for pathogenic microorganisms that cause nosocomial infections. Nosocomial pathogens may survive on hard surfaces, viable for long periods of time depending on the type of microorganism and environmental conditions [1, 1]. Preventive interventions to reduce the levels of microbial contamination decrease nosocomial infections in hospital settings [27]. There is an urgent need for new approaches in the field of infection control and the antimicrobial activity of copper could provide such an approach for the current measures to prevent dissemination of nosocomial MDR strains in hospitals. In an attempt to mimic in vivo conditions, various laboratory-based studies have used numerous methods to show the efficacy of copper against pathogenic organisms Fig. 1 Inactivation of six A. baumannii (a), four P. aeruginosa (b), and four MRSA (c) isolates on copper alloy (Cu63 %), copper (Cu99 %) and SS. Results are shown as mean number of viable colonies against time intervals (a) (b) (c)

4 294 O. Koseoglu Eser et al.: Antimicrobial Activity of Copper Alloys Against Invasive Table 1 Bactericidal and bacteriostatic effects of Cu63, Cu99, and SS against microorganisms in each time interval Surface material Acinetobacter baumannii Cu Cu a a a a a SS Pseudomonas aeruginosa Cu a a a b Cu a b b b b SS a a Staphylococcus aureus Cu Cu b -3.2 b -3.2 b -3.2 b -3.2 b SS a Bacteriostatic b Bactericidal effect [13, 22]. There are mainly two wet and dry inoculation techniques used in studies and it has been shown that temperature, humidity, the copper content of alloys and the dry technique increase the efficacy of copper contact killing [8]. We have decided to use the droplet technique (dry method) in our study, since this technique has been proved to show more reproducible results than the other method. According to our results, 99 % copper exhibited the highest reduction of microorganisms and the significance was particularly apparent at, 2, and 4 h time intervals. The bactericidal effect of 99 % copper was shorter than the other studies that have evaluated multidrug-resistant bacteria [2, 7, 22, 23]. Two separate studies, Warnes and Keevil [26] and Santo et al. [2], recently published that killing of bacteria on a dry copper surface can be as short as 2 1 min. This situation is in agreement with our data but the tested microorganisms (enterococci and nonpathogenic Gram positive bacteria) were different from the strains used in our study. Our results show that organisms such as ESBL producing MDR A. baumannii, methicillinresistant S. aureus and MDR P. aeruginosa were killed relatively quickly on the 99 % copper surface. All A. baumannii, P. aeruginosa and MRSA isolates were nearly killed within 6 min, in other words the antimicrobial resistance patterns and resistance genes of the isolates did not appear to have an effect on the antimicrobial activity of 99 % copper. Many of the previous studies have examined pure copper as well as copper-based alloys and in all of these studies the pure copper was shown to be the most effective contact material [7, 9, 15]. The problem is the question of the copper content of the environmental surfaces since 99 % copper is a flexible metal and therefore likely to wear faster than a copper alloy such as brass or zinc which are hard and more durable metals, so in some cases copper alloys can be used like copper zinc or brass [14, 17, 25]. Copper is the only hard surface metal that has received approval by the EPA and this approval has now been given to nearly 3 different copper alloys. This is an important step for the protection of public health and hospital environment issues [4]. According to our results, 63 % copper alloy had compatible results for P. aeruginosa. This alloy had a bacteriostatic effect starting from the 2 h period and had reached a bactericidal effect in the 24 h period against our P. aeruginosa isolates. There was no statistically significant difference between the three materials for all microorganisms in 6 and 24 h but 63 % copper and stainless steel surfaces exhibited a and log1 cfu/cm 2 change in initial inoculum of P. aeruginosa at 24 h. The results have shown the non-surviving characteristics of this microorganism on dry surfaces. This data might be an alternative in case of consideration of better strength for contact surfaces of hospital environments. The results of this study expanded the results of preliminary observations concerning the bactericidal efficacy of 99 % copper and validated the previous reports [22]. Our results have two main differences from previous studies. The main difference of the study concerned the duration of the killing time interval of 99 % Cu. According to our results, it was almost zero time for such problematic MDR nosocomial bacterial strains. The second important result concerned the bactericidal effect of 63 %Cu in P. aeruginosa isolates. This data might be important with regard to the usage of a more durable and effective material in hospital settings. The strong efficacy of copper alloys against several types of invasive MDR pathogens indicates that this material should be subjected to evaluation in hospital settings, especially in intensive care units. The bactericidal efficacy of copper as part of a contact surface has been extensively studied but the clinical impact of this efficacy is unclear. Limited hospital trials have demonstrated that significant reductions in nosocomial infections were observed when copper alloys were used in hospital settings [21]. Further to the laboratory and clinical research results, there is now a great interest in producing objectives that will be used by manufacturers in high nosocomial areas like ICUs and medical wards [4]. The current study has some limitations. We only demonstrated the in vitro efficacy of copper alloys and stainless steel against the pathogens used. We could not evaluate the efficacy of copper alloys under indoor conditions in hospital settings but the isolates used were all nosocomial invasive strains and had the characteristics of multidrug-resistant pathogens, which can provide evidence for the serious problems in ICUs and other hospital areas.

5 O. Koseoglu Eser et al.: Antimicrobial Activity of Copper Alloys Against Invasive 295 Other nosocomial pathogens such as vancomycin-resistant enterococci or Clostridium difficile could be included in the study but these pathogens are still not a frequent cause of hospital-acquired infections in our country. As a result, 99 % copper reduced the number of bacteria used significantly and produced a bactericidal effect and was more effective than 63 % copper alloy and stainless steel. The data suggests that use of copper as a surface material could aid in eliminating the environmental reservoir of important invasive nosocomial pathogens and may reduce the opportunity for the cross-transfer of these superbugs in the hospital environment. However, it is still worth saying that the decision for the implementation of copper contact in hospital settings should be given after further clinical trials. Acknowledgments This study was presented in the 114th General Meeting of the American Society for Microbiology, May 17 2, 214, Boston, Massachusetts, USA. We would like to thank Selçuk Korkmaz (Hacettepe University, Department of Biostatistics) for interpreting the statistical analysis of the data and Tülay Özçelik for technical assistance. References 1. Borkow G, Monk A (212) Fighting nosocomial infections with biocidal non-intrusive hard and soft surfaces. World J Infect Dis 2: Cervantes HI, Alvarez JA, Munoz JM, Arreguin V, Mosqueda JL, Macias AE (213) Antimicrobial activity of copper against organisms in aqueous solution: a case for copper-based water pipelines in hospitals? Am J Infect Control 41:e115 e Clinical and Laboratory Standards Institute (CLSI) (213) Performance standards for antimicrobial susceptibility testing; twenty-third informational supplement CLSI document M1- S23, Wayne, PA, USA 4. 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