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[Akiko Yamamoto](https://orcid.org/0000-0002-9182-4886), Shinji Tanaka, Keiichiro Ohishi

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[Quantitative Evaluation of Nucleic Acid Degradability of Copper Alloy Surfaces and Its Correlation to Antibacterial Activity](https://mdr.nims.go.jp/datasets/6bfd892c-72e0-4618-9f92-eafce99f2b03)

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Quantitative Evaluation of Nucleic Acid Degradability of Copper Alloy Surfaces and Its Correlation to Antibacterial ActivityantibioticsArticleQuantitative Evaluation of Nucleic Acid Degradability ofCopper Alloy Surfaces and Its Correlation toAntibacterial ActivityAkiko Yamamoto 1,*, Shinji Tanaka 2 and Keiichiro Ohishi 2�����������������Citation: Yamamoto, A.; Tanaka, S.;Ohishi, K. Quantitative Evaluation ofNucleic Acid Degradability of CopperAlloy Surfaces and Its Correlation toAntibacterial Activity. Antibiotics2021, 10, 1439. https://doi.org/10.3390/antibiotics10121439Academic Editors: Marc Maresca,Catherine Lefay andVincent HumblotReceived: 26 October 2021Accepted: 23 November 2021Published: 24 November 2021Publisher’s Note: MDPI stays neutralwith regard to jurisdictional claims inpublished maps and institutional affil-iations.Copyright: © 2021 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).1 Research Center for Functional Materials, National Institute for Materials Science, 1-1 Namiki,Tsukuba 305-0044, Japan2 Research & Development Department, Sambo Plant, Mitsubishi Materials Corporation, Sambo-cho 8-374,Sakai-ku, Sakai-shi 590-0906, Japan; tanakas@mmc.co.jp (S.T.); oishik@mmc.co.jp (K.O.)* Correspondence: YAMAMOTO.Akiko@nims.go.jpAbstract: Copper (Cu) and its alloys have bactericidal activity known as “contact killing” with degra-dation of nucleic acids inside the bacteria, which is beneficial to inhibit horizontal gene transfer (HGF).In order to understand the nucleic acid degradability of Cu and its alloy surfaces, we developed anew in vitro method to quantitatively evaluate it by a swab method under a “dry” condition andcompared it with that of commercially available antibacterial materials such as antibacterial stainlesssteel, pure silver, and antibacterial resins. As a result, only Cu and its alloys showed continuousdegradation of nucleic acids for up to 6 h of contact time. The nucleic acid degradability levels of theCu alloys and other antibacterial materials correlate to their antibacterial activities evaluated by a filmmethod referring to JIS Z 2801:2012 for Gram-negative (Escherichia coli) and Gram-positive (Staphy-lococcus aureus) bacteria. Nucleic acid degradation by copper (I) and (II) chlorides was confirmedat the ranges over 10 mM and 1–20 mM, respectively, suggesting that the copper ion release maybe responsible for the degradation of the nucleic acids on Cu and its alloy surfaces. In conclusion,the higher Cu content in the alloys gave higher nucleic acid degradability and higher antibacterialactivities.Keywords: antimicrobial activities; antibacterial test; copper and copper alloys; DNA degradability;a swab method1. IntroductionAntimicrobial resistance (AMR) in bacteria has become a global threat, and deathsattributable to AMR are estimated to reach over 10 million people by 2050 [1]. Unnecessaryand excessive antimicrobial use should be avoided worldwide, suggesting the necessity ofnew sanitation methods without antimicrobials. Contaminated touch surfaces in hospitalrooms play an important role in the transmission of healthcare-associated pathogens [2],thus, it is important to control the number of viable bacteria (bioburden) on touch surfaces,since methicillin-resistant Staphylococcus aureus (MRSA), one of the causes of healthcare-associated infection (HAI) in hospitals, can survive for months with infectivity on generaltouch surfaces of resin and stainless steel [3]. Under these circumstances, metal copperand its alloys have attracted attention in healthcare-related fields due to their excellentantimicrobial activity. Much research has been carried out to conclude that microorganisms,including Gram-negative and Gram-positive bacteria, fungi, spores, yeasts, and viruses,are rapidly killed on copper and its alloy surfaces, which is described by the term “contactkilling” [4–6]. Prolonged exposure of microorganisms to copper and its alloy surfacesresulted in no recovery of viable microorganisms. Therefore, copper and its alloys contain-ing copper with over 60% in their composition were registered at the US EnvironmentalProtection Agency as the first solid antimicrobial material [6]. Introduction of copper andAntibiotics 2021, 10, 1439. https://doi.org/10.3390/antibiotics10121439 https://www.mdpi.com/journal/antibioticshttps://www.mdpi.com/journal/antibioticshttps://www.mdpi.comhttps://doi.org/10.3390/antibiotics10121439https://doi.org/10.3390/antibiotics10121439https://creativecommons.org/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://doi.org/10.3390/antibiotics10121439https://www.mdpi.com/journal/antibioticshttps://www.mdpi.com/article/10.3390/antibiotics10121439?type=check_update&version=2Antibiotics 2021, 10, 1439 2 of 15its alloys to the touch surfaces such as door handles, washroom fixtures, and bed railshave been carried out in hospitals and other healthcare-related institutes to achieve thereduction of bioburden and HAI [7–10]. Generally, higher antibacterial activity is obtainedwith copper alloys having higher copper contents [4,11]. However, pure copper easilychanges its surface color by oxidation; therefore, copper alloys are practical candidates toapply touch surfaces.Another feature of the antibacterial activity of copper and its alloys is that only afew copper-resistant bacteria have been reported. This may be related to the degradationof bacterial nucleic acids observed on copper and its alloy surfaces [12–18]. Bacteriahave the ability of horizontal gene transfer (HGT), which can occur on touch surfaces.By HGT, the antibiotic-resistant gene can be transferred to another bacterium that neverencounters a corresponding antibiotic; this process may contribute to the generation ofmulti-antibiotic-resistant bacteria [15]. HGT can occur even with remaining nucleic acidsfrom dead bacteria, therefore, the degradability of bacterial nucleic acids on the materialsurface is one of the essential properties for the materials applied to these touch surfaces.For its further utilization and optimization, it is important to appropriately evaluate thenucleic acids degradability of materials as well as to investigate its mechanism. In order toevaluate the fragmentation of bacterial genes, electrophoresis of nucleic acids extractedfrom the bacteria applied onto the material surface is mostly performed [12,14–18], buta quantitative evaluation is difficult since bacterial numbers will change at the materialsurface depending on its antibacterial activity. There is no quantitative evaluation methodof a material’s ability to degrade nucleic acids on its surface.In this study, we developed a new in vitro method to evaluate the nucleic acid degrad-ability of the material surface. Pure copper, three kinds of copper alloys (CLEANBRASS®,CLEANBRIGHT®, and C6932), and commercially available antibacterial materials suchas resins (X and Y) and an antibacterial stainless steel (NSSAM3 [19], abbreviated asABSS) were employed. Pure silver (Ag) is also tested as a reference material. Nucleicacid degradability of these material surfaces was evaluated and its correlation to theirantibacterial activity for Gram-negative (Escherichia coli) and Gram-positive (Staphylococcusaureus) bacteria was examined.2. Results2.1. Nucleic Acid Degradability of Testing MaterialsIn our developed method, a 1 µL portion of nucleic acid solution was directly spreadonto the material surface, which dries within 5 min, simulating the situation of an airbornedroplet. After a certain period of contact time, the material surface was wiped with apolyester swab wetted by a small portion (20 µL) of an extract solvent (0.03% sodiumdodecyl sulphate; SDS). Then, the remaining nucleic acid in the swab was further extractedinto the solvent and quantified using a fluorescent die, acridine orange (AO), a nucleic acidintercalater. Since the bacterial cells are not involved, the nucleic acid degradability of thematerial can be evaluated independently from the bacterial growth or death on its surface.This kind of swabbing method is widely employed to monitor bioburden in a hospitalenvironment [8], but not for the evaluation of nucleic acid degradability. A non-ionicsurfactant was added to the nucleic acid solution, enabling us to control its spreadabilityand drying time on the different material surfaces.The results of nucleic acid degradation assay are shown in Figure 1. The averagerecovery rates of nucleic acids from the glass surface were 70–75% at time “0” (5 min), whichsuggests the loss of the nucleic acid samples due to swabbing and extracting processesin this assay. However, the recovery rates from the pure copper (C1020) were 14.2% fordouble-stranded deoxyribonucleic acids (dsDNA) and 4.8% for single-stranded DNA andribonucleic acids (ssDNA + RNA) even at time “0”. The recovery rates from copperalloys were 8–27%, except CLEANBRIGHT® (CBRI). The recovery rates from CBRI were44% for both of dsDNA and ssDNA + RNA at time “0”, but they decreased to 15.1 and6.0%, respectively, with an increase in contact time. In the cases of ABSS, Ag, and resins,Antibiotics 2021, 10, 1439 3 of 15the recovery rates of nucleic acids stayed 40–75% regardless of their contact time. Themetallic silver had a relatively low recovery rate of dsDNA as ~40% than other non-coppermaterials, which was closer to that of CBRI, but it was stable through the contact time. Therecovery rate of nucleic acids may be influenced by their adsorption kinetics to differentmaterial surfaces, therefore, the time-dependent decrease in the recovery rate is consideredas a good indicator of the material’s nucleic acids degradability.Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 15  rates from CBRI were 44% for both of dsDNA and ssDNA + RNA at time “0”, but they decreased to 15.1 and 6.0%, respectively, with an increase in contact time. In the cases of ABSS, Ag, and resins, the recovery rates of nucleic acids stayed 40–75% regardless of their contact time. The metallic silver had a relatively low recovery rate of dsDNA as ~40% than other non-copper materials, which was closer to that of CBRI, but it was sta-ble through the contact time. The recovery rate of nucleic acids may be influenced by their adsorption kinetics to different material surfaces, therefore, the time-dependent decrease in the recovery rate is considered as a good indicator of the material’s nucleic acids degradability. Figure 2 is the plot of the nucleic acid recovery rates at time “0” against those at 6 h. It clearly categorizes testing materials into two groups; materials reduced the recovery rates with the increase in the contact time, and others had constant recovery rates re-gardless of the contact time. The first group includes copper and its alloys.  Figure 1. Recovery rates of (a) double-stranded deoxyribonucleic acids (dsDNAs) and (b) sin-gle-stranded (ss) DNA and ribonucleic acid (RNA) contacted on the material surface after a certain period of time (mean ± standard deviation). 0204060801001200 1 3 6dsDNA recovery rate(%)Contact time (h)control C1020 CBRA CBRI C6932NSSAM3 Ag resin I resin T(a)0204060801001200 1 3 6ssDNA+RNArecovery rate(%)Contact time (h)control C1020 CBRA CBRI C6932NSSAM3 Ag resin I resin TX YX Y(b)ABSSABSSFigure 1. Recovery rates of (a) double-stranded deoxyribonucleic acids (dsDNAs) and (b) single-stranded (ss) DNA and ribonucleic acid (RNA) contacted on the material surface after a certainperiod of time (mean ± standard deviation).Figure 2 is the plot of the nucleic acid recovery rates at time “0” against those at 6 h. Itclearly categorizes testing materials into two groups; materials reduced the recovery rateswith the increase in the contact time, and others had constant recovery rates regardless ofthe contact time. The first group includes copper and its alloys.Figure 3 plotted the nucleic acid recovery rates against the copper content in the alloycomposition, indicating the alloy with the higher copper content gives the lower recoveryrates, which means the higher nucleic acid degradability. It also suggests that the rapidnucleic acid degradability may be a unique property of copper and its alloys, not achievedby other commercially available antibacterial materials such as ABSS and resins.Antibiotics 2021, 10, 1439 4 of 151   0204060801000 50 100Nucleic acid recovery rate at 6h(%)Nucleic acid recovery rate at 0h(%)dsDNAssDNA+RNAFigure 2. Correlation of nucleic acid recovery rates at 0 h and 6 h. The dotted line indicates nucleicacid recovery rate at 0 h equals to that at 6 h (y = x). Dashed circle indicates the results of copper andits alloys.Antibiotics 2021, 10, x FOR PEER REVIEW 4 of 15   Figure 2. Correlation of nucleic acid recovery rates at 0 h and 6 h. The dotted line indicates nucleic acid recovery rate at 0 h equals to that at 6 h (y = x). Dashed circle indicates the results of copper and its alloys. Figure 3 plotted the nucleic acid recovery rates against the copper content in the al-loy composition, indicating the alloy with the higher copper content gives the lower re-covery rates, which means the higher nucleic acid degradability. It also suggests that the rapid nucleic acid degradability may be a unique property of copper and its alloys, not achieved by other commercially available antibacterial materials such as ABSS and res-ins.  Figure 3. Dependence of nucleic acid recovery rate at 6 h on the copper content in the alloy com-position (mean ± standard deviation). 2.2. Nucleic Acid Degradation by Copper Salts In regard to the antibacterial activities of the copper and its alloys, the involvement of released copper ions from the metal surface is suggested as one of the contact killing mechanisms by its uptake and accumulation into the bacterial cells [4,20]. The nucleic acid degradation by copper salts was investigated in a similar condition and shown in Figure 4. Copper (I) and (II) chlorides, which were employed as the sources of copper (I) and (II) ions, both showed the rapid degradation of nucleic acid samples within 5 min with different concentration ranges; >1 mM for copper (II) chlorides whereas >10 mM for copper (I) salts. 0204060801000 50 100Nucleic acid recovery rate at 6h(%)Nucleic acid recovery rate at 0h(%)dsDNAssDNA+RNAy = -17.91ln(x) + 85.221R² = 0.9928y = -24.42ln(x) + 107.3R² = 0.98740204060801001 10 100Nucleic acid recovery rate at 6h(%)Cu content in composition (wt.%)dsDNA_6hssDNA+RNA_6hFigure 3. Dependence of nucleic acid recovery rate at 6 h on the copper content in the alloy composi-tion (mean ± standard deviation).2.2. Nucleic Acid Degradation by Copper SaltsIn regard to the antibacterial activities of the copper and its alloys, the involvementof released copper ions from the metal surface is suggested as one of the contact killingmechanisms by its uptake and accumulation into the bacterial cells [4,20]. The nucleicacid degradation by copper salts was investigated in a similar condition and shown inFigure 4. Copper (I) and (II) chlorides, which were employed as the sources of copper (I)and (II) ions, both showed the rapid degradation of nucleic acid samples within 5 minwith different concentration ranges; >1 mM for copper (II) chlorides whereas >10 mM forcopper (I) salts.Antibiotics 2021, 10, x FOR PEER REVIEW 5 of 15   Figure 4. Nucleic acid remaining rates measured at 5 min after adding a certain concentration of CuCl or CuCl2 (mean ± standard deviation). The copper ion release from the pure copper (C1020) during the nucleic acid deg-radation assay was analyzed by a colorimetric method, as shown in Figure 5. Since this assay was carried out under “dry” condition simulating the airborne droplets, the nu-cleic acid portion placed on the C1020 surface was dried within 5 min in the ambient conditions, and stayed dry during the contact time. The concentrations of the released copper ions were stable at 44–62 µM regardless of the contact time (0–6 h).  Figure 5. Copper ion release from C1020 under the same condition to nucleic acid degradation as-say (mean ± standard deviation). 2.3. Antibacterial Activity of Testing Materials In order to investigate the correlation between the nucleic acid degradability of testing materials with their antibacterial activities, antibacterial tests were carried out by a film method referring to JIS Z2801:2012 (ISO 22196) using E. coli and S. aureus. The re-sults are shown in Figure 6 as the cell viability plotted against the contact time. Based on the results, testing materials are classified into three groups; (A) copper and its alloys, (B) silver and antibacterial stainless steel, and (C) commercially available antibacterial resins for both the Gram-negative and Gram-positive bacteria. For group A materials, the number of viable cells rapidly decreased to 1/10 of the inoculated cells within 20 min. The decreasing rate of the bacterial cells depends on the type of copper alloys, and was the largest on C1020, followed by C6932 and CLEANBRASS® (CBRA), and then, by CBRI. In the case of C1020, the number of viable cells decreased to 1/100 within 10 min. For group B, the number of the viable cells decreased less than 1/10 at 24 h, but not at 30 or 120 min. For group C, the number of the viable cells was similar to that of the control material, glass after 24 h. 0%50%100%150%0.1 1 10 100Nucleic acid remaining rate(%)concentration (mM)CuCl_dsDNACuCl_ssDNA+RNACuCl2_dsDNACuCl2_ssDNA+RNA1501005001101000 100 200 300 400Cu ion concentration (µM)contact time (min)C1020glassFigure 4. Nucleic acid remaining rates measured at 5 min after adding a certain concentration ofCuCl or CuCl2 (mean ± standard deviation).Antibiotics 2021, 10, 1439 5 of 15The copper ion release from the pure copper (C1020) during the nucleic acid degrada-tion assay was analyzed by a colorimetric method, as shown in Figure 5. Since this assaywas carried out under “dry” condition simulating the airborne droplets, the nucleic acidportion placed on the C1020 surface was dried within 5 min in the ambient conditions, andstayed dry during the contact time. The concentrations of the released copper ions werestable at 44–62 µM regardless of the contact time (0–6 h).Antibiotics 2021, 10, x FOR PEER REVIEW 5 of 15   Figure 4. Nucleic acid remaining rates measured at 5 min after adding a certain concentration of CuCl or CuCl2 (mean ± standard deviation). The copper ion release from the pure copper (C1020) during the nucleic acid deg-radation assay was analyzed by a colorimetric method, as shown in Figure 5. Since this assay was carried out under “dry” condition simulating the airborne droplets, the nu-cleic acid portion placed on the C1020 surface was dried within 5 min in the ambient conditions, and stayed dry during the contact time. The concentrations of the released copper ions were stable at 44–62 µM regardless of the contact time (0–6 h).  Figure 5. Copper ion release from C1020 under the same condition to nucleic acid degradation as-say (mean ± standard deviation). 2.3. Antibacterial Activity of Testing Materials In order to investigate the correlation between the nucleic acid degradability of testing materials with their antibacterial activities, antibacterial tests were carried out by a film method referring to JIS Z2801:2012 (ISO 22196) using E. coli and S. aureus. The re-sults are shown in Figure 6 as the cell viability plotted against the contact time. Based on the results, testing materials are classified into three groups; (A) copper and its alloys, (B) silver and antibacterial stainless steel, and (C) commercially available antibacterial resins for both the Gram-negative and Gram-positive bacteria. For group A materials, the number of viable cells rapidly decreased to 1/10 of the inoculated cells within 20 min. The decreasing rate of the bacterial cells depends on the type of copper alloys, and was the largest on C1020, followed by C6932 and CLEANBRASS® (CBRA), and then, by CBRI. In the case of C1020, the number of viable cells decreased to 1/100 within 10 min. For group B, the number of the viable cells decreased less than 1/10 at 24 h, but not at 30 or 120 min. For group C, the number of the viable cells was similar to that of the control material, glass after 24 h. 0%50%100%150%0.1 1 10 100Nucleic acid remaining rate(%)concentration (mM)CuCl_dsDNACuCl_ssDNA+RNACuCl2_dsDNACuCl2_ssDNA+RNA1501005001101000 100 200 300 400Cu ion concentration (µM)contact time (min)C1020glassFigure 5. Copper ion release from C1020 under the same condition to nucleic acid degradation assay(mean ± standard deviation).2.3. Antibacterial Activity of Testing MaterialsIn order to investigate the correlation between the nucleic acid degradability of testingmaterials with their antibacterial activities, antibacterial tests were carried out by a filmmethod referring to JIS Z2801:2012 (ISO 22196) using E. coli and S. aureus. The results areshown in Figure 6 as the cell viability plotted against the contact time. Based on the results,testing materials are classified into three groups; (A) copper and its alloys, (B) silver andantibacterial stainless steel, and (C) commercially available antibacterial resins for both theGram-negative and Gram-positive bacteria. For group A materials, the number of viablecells rapidly decreased to 1/10 of the inoculated cells within 20 min. The decreasing rateof the bacterial cells depends on the type of copper alloys, and was the largest on C1020,followed by C6932 and CLEANBRASS® (CBRA), and then, by CBRI. In the case of C1020,the number of viable cells decreased to 1/100 within 10 min. For group B, the number ofthe viable cells decreased less than 1/10 at 24 h, but not at 30 or 120 min. For group C, thenumber of the viable cells was similar to that of the control material, glass after 24 h.Antibiotics 2021, 10, x FOR PEER REVIEW 6 of 15   Figure 6. Antimicrobial activities of materials evaluated by the film method referring to JIS Z2801:2012 (ISO 22196) against Escherichia coli (a) and Staphylococcus aureus (b) (mean ± standard deviation). Based on the results in Figure 6, the time to decrease the viable cells to 1/10 or 1/100 of the inoculated number (T1/10 or T1/100) was calculated by the probit method and shown in Table 1. These parameters clearly identify the difference in the antibacterial activities of testing materials as three groups; group A with T1/10 of ~20 min, group B with T1/10 of ~500 min, and group C with T1/10 of >1000 min, that corresponds to the copper and the copper alloys, ABSS and Ag, and the antibacterial resins, respectively. Table 1. Time to reduce the number of viable cells to 1/10 and 1/100 of the inoculated number of cells on the material surface (T1/10 and T1/100, min). C1020 C6932 CBRA CBRI ABSS Ag Glass Resin X Resin Y E.coli T1/10 6.27 9.97 10.2 15.7 383 575 1230 1830 2670 T1/100 9.33 24.6 23.4 34.6 1490 1990 4060 10,500 20,400 S.aureus T1/10 5.30 10.3 9.89 15.8 504 530 1120 1000 1850 T1/100 9.86 25.0 23.8 30.7 2030 1780 5280 4630 10,900 Antimicrobial activity was evaluated by the film method referring to JIS Z2801:2012 (ISO 22196) except culture media (0.9%NaCl) and extract solution (0.9% NaCl + 0.1 mM EDTA-1Na). T1/10 and T1/100 were calculated by the probit method. CBRA: CLEANBRASS®, CBRI: CLEANBRIGHT®, ABSS: antibacterial stainless steel [19], resins X and Y were cut from the commercially available antimicrobial toilet seats and lids. Figure 7 is the plot of the T1/10 for E.coli against T1/10 for S.aureus, confirming the good correlation between Gram-negative and Gram-positive bacteria. In this figure, the classification of 3 groups (A–C) is identified as circled, indicating the higher bacterial ac-tivities of group A than others. Figure 8 plotted the T1/10 for both kinds of bacteria against the copper content in the alloy composition. This figure suggests the higher copper con-tent in the alloy contributes to the higher antibacterial activities. 0.0010.010.11101 10 100 1000 10000Cell viability(%)Contact time(min)C1020 CBRI CBRAC6932 ABSS Agglass resin T resin Icontrol XY(a)10,0000.0010.010.11101 10 100 1000 10000Cell viability(%)Contact time(min)C1020 CBRI CBRAC6932 ABSS Agglass resin T resin Icontrol XY(b)10,000Figure 6. Antimicrobial activities of materials evaluated by the film method referring to JIS Z2801:2012 (ISO 22196) againstEscherichia coli (a) and Staphylococcus aureus (b) (mean ± standard deviation).Antibiotics 2021, 10, 1439 6 of 15Based on the results in Figure 6, the time to decrease the viable cells to 1/10 or 1/100of the inoculated number (T1/10 or T1/100) was calculated by the probit method and shownin Table 1. These parameters clearly identify the difference in the antibacterial activities oftesting materials as three groups; group A with T1/10 of ~20 min, group B with T1/10 of~500 min, and group C with T1/10 of >1000 min, that corresponds to the copper and thecopper alloys, ABSS and Ag, and the antibacterial resins, respectively.Table 1. Time to reduce the number of viable cells to 1/10 and 1/100 of the inoculated number of cells on the materialsurface (T1/10 and T1/100, min).C1020 C6932 CBRA CBRI ABSS Ag Glass Resin X Resin YE.coliT1/10 6.27 9.97 10.2 15.7 383 575 1230 1830 2670T1/100 9.33 24.6 23.4 34.6 1490 1990 4060 10,500 20,400S.aureusT1/10 5.30 10.3 9.89 15.8 504 530 1120 1000 1850T1/100 9.86 25.0 23.8 30.7 2030 1780 5280 4630 10,900Antimicrobial activity was evaluated by the film method referring to JIS Z2801:2012 (ISO 22196) except culture media (0.9%NaCl) andextract solution (0.9% NaCl + 0.1 mM EDTA-1Na). T1/10 and T1/100 were calculated by the probit method. CBRA: CLEANBRASS®, CBRI:CLEANBRIGHT®, ABSS: antibacterial stainless steel [19], resins X and Y were cut from the commercially available antimicrobial toilet seatsand lids.Figure 7 is the plot of the T1/10 for E.coli against T1/10 for S.aureus, confirming thegood correlation between Gram-negative and Gram-positive bacteria. In this figure, theclassification of 3 groups (A–C) is identified as circled, indicating the higher bacterialactivities of group A than others. Figure 8 plotted the T1/10 for both kinds of bacteriaagainst the copper content in the alloy composition. This figure suggests the higher coppercontent in the alloy contributes to the higher antibacterial activities.Antibiotics 2021, 10, x FOR PEER REVIEW 7 of 15   Figure 7. Correlation of the time to reduce the number of viable cells 1/10 of the initial number of cells (T1/10) for testing materials against E. coli and S.aureus. The classification of 3 groups (A–C) is identified as circled.  Figure 8. Dependence of T1/10 on the copper content in the alloy composition. 2.4. Correlation between Nucleic Acid Degradability and Antibacterial Activity of Testing Materials Figure 9 shows the plots of T1/10 for both E.coli and S.aureus against dsDNA and ssDNA + RNA recovery rates after 6 h of contact time, respectively. It clearly shows the trend that the material having low T1/10 has a low nucleic acid recovery rate. Even among the copper and its alloys, the higher copper content in the alloy composition resulted in the higher antibacterial activity and nucleic acid degradability.  y = 1.0903x0.9553R² = 0.99241101001000100001 10 100 1000 10000T 1/10for S.aureus(min)T1/10 for E.coli(min)CAB10,00010,000y = 2442.5x-1.283R² = 0.9992y = 3527.2x-1.38R² = 0.995911010010001 10 100T 1/10(min)Cu content in composition (wt.%) E.coliS.aureusy = 6.5703e0.0839xR² = 0.9087y = 8.5439e0.066xR² = 0.96251101001000100000 50 100T 1/10(min)Nucleic acid recovery rate(%)E.coli_dsDNA_6hS.aureus_ssDNA+RNA_6h10,000Figure 7. Correlation of the time to reduce the number of viable cells 1/10 of the initial number ofcells (T1/10) for testing materials against E. coli and S.aureus. The classification of 3 groups (A–C) isidentified as circled.Antibiotics 2021, 10, x FOR PEER REVIEW 7 of 15   Figure 7. Correlation of the time to reduce the number of viable cells 1/10 of the initial number of cells (T1/10) for testing materials against E. coli and S.aureus. The classification of 3 groups (A–C) is identified as circled.  Figure 8. Dependence of T1/10 on the copper content in the alloy composition. 2.4. Correlation between Nucleic Acid Degradability and Antibacterial Activity of Testing Materials Figure 9 shows the plots of T1/10 for both E.coli and S.aureus against dsDNA and ssDNA + RNA recovery rates after 6 h of contact time, respectively. It clearly shows the trend that the material having low T1/10 has a low nucleic acid recovery rate. Even among the copper and its alloys, the higher copper content in the alloy composition resulted in the higher antibacterial activity and nucleic acid degradability.  y = 1.0903x0.9553R² = 0.99241101001000100001 10 100 1000 10000T 1/10for S.aureus(min)T1/10 for E.coli(min)CAB10,00010,000y = 2442.5x-1.283R² = 0.9992y = 3527.2x-1.38R² = 0.995911010010001 10 100T 1/10(min)Cu content in composition (wt.%) E.coliS.aureusy = 6.5703e0.0839xR² = 0.9087y = 8.5439e0.066xR² = 0.96251101001000100000 50 100T 1/10(min)Nucleic acid recovery rate(%)E.coli_dsDNA_6hS.aureus_ssDNA+RNA_6h10,000Figure 8. Dependence of T1/10 on the copper content in the alloy composition.Antibiotics 2021, 10, 1439 7 of 152.4. Correlation between Nucleic Acid Degradability and Antibacterial Activity of TestingMaterialsFigure 9 shows the plots of T1/10 for both E.coli and S.aureus against dsDNA andssDNA + RNA recovery rates after 6 h of contact time, respectively. It clearly shows thetrend that the material having low T1/10 has a low nucleic acid recovery rate. Even amongthe copper and its alloys, the higher copper content in the alloy composition resulted in thehigher antibacterial activity and nucleic acid degradability.Antibiotics 2021, 10, x FOR PEER REVIEW 7 of 15   Figure 7. Correlation of the time to reduce the number of viable cells 1/10 of the initial number of cells (T1/10) for testing materials against E. coli and S.aureus. The classification of 3 groups (A–C) is identified as circled.  Figure 8. Dependence of T1/10 on the copper content in the alloy composition. 2.4. Correlation between Nucleic Acid Degradability and Antibacterial Activity of Testing Materials Figure 9 shows the plots of T1/10 for both E.coli and S.aureus against dsDNA and ssDNA + RNA recovery rates after 6 h of contact time, respectively. It clearly shows the trend that the material having low T1/10 has a low nucleic acid recovery rate. Even among the copper and its alloys, the higher copper content in the alloy composition resulted in the higher antibacterial activity and nucleic acid degradability.  y = 1.0903x0.9553R² = 0.99241101001000100001 10 100 1000 10000T 1/10for S.aureus(min)T1/10 for E.coli(min)CAB10,00010,000y = 2442.5x-1.283R² = 0.9992y = 3527.2x-1.38R² = 0.995911010010001 10 100T 1/10(min)Cu content in composition (wt.%) E.coliS.aureusy = 6.5703e0.0839xR² = 0.9087y = 8.5439e0.066xR² = 0.96251101001000100000 50 100T 1/10(min)Nucleic acid recovery rate(%)E.coli_dsDNA_6hS.aureus_ssDNA+RNA_6h10,000Figure 9. Correlation between nucleic acid recovery rate at 6 h and the antimicrobial activity, T1/10.3. Discussion3.1. Antibacterial Activity of Copper, Copper Alloys, and Other Antibacterial MaterialsAntibacterial activities of the materials were evaluated by contacting bacterial cellsuspensions to the material surfaces for a certain time and counting the number of recoveredcells from the testing materials. The most popular method to count the number of recoveredcells is an agar plate cultivation method using 10-fold serial dilutions. This method hashigh sensitivity and can be performed without any special equipment, but it requires arelatively long time, such as 40–48 h, for the formation of bacterial colonies [21]. In thisstudy, the 5-Cyano-2,3-ditolyl-2H-tetrazolium chloride (CTC) method [22,23] is employedto quantify the viable cells (with respiratory activity) under fluorescent measurement. Thismethod has the advantage of counting the viable cells fairly quickly, such as 30 min ofincubation with CTC, but it has a disadvantage of relatively low sensitivity (detection limit).Ideally, the agar plate cultivation method can detect a single cell if it can grow and forma colony, but the detection limit of the CTC method is about ~4000 cells at the conditionemployed in this study.As shown in Figure 6, testing materials are classified into three groups based on theirantibacterial activities, indicating the highest antibacterial activity of copper and copperalloys, followed by non-copper antibacterial metallic materials, and then, antibacterialresins. In the case of C1020, the number of viable cells decreased to 1/100 within 10 min.This rapid decrease in bacterial cells on the C1020 surface agrees with the previous reportsusing Escherichia coli (W3110 [13] and NCTC13441 [15]) and those using MRSA [18,24] eventhough they use slightly different cell strains with the larger inoculated number of cells. Italso agreed with these reports [15,18,24] that the antibacterial activity of pure copper washigher than those of copper alloys. Generally, the higher copper content in the alloy givesthe higher antibacterial activities [4,11,15,18,24,25], which is also confirmed in the presentstudy. These facts suggest that the viable cell counts by CTC can give similar results tothose by the agar plate cultivation method and that the CTC method is useful to obtain theantibacterial activities of various materials in a relatively short experimental time.Though much research has investigated the antibacterial activity of copper and itsalloys, few reports compare their antibacterial activities with those of commercially avail-Antibiotics 2021, 10, 1439 8 of 15able antibacterial materials. Antibacterial stainless steel and resins employed in the presentstudy are authenticated by the antibacterial tests following JIS Z2801:2012 [26], equivalentto ISO 22196 [21]. In this standard, antibacterial activity is defined by the comparison of theviable number of cells between the target material and that without antibacterial treatmentafter 24 h of contact time. The antibacterial material is required to have the recoveredcells two orders of magnitude less than those from the non-treated material [26]. On thisdefinition, it does not mean that the recovered cells from the target material are less thanthe inoculated cells. In the present study, the number of the bacterial cells recovered fromABSS after 24 h of contact decreased almost 1/100 of the inoculated number, suggesting thebactericidal activity on its surface. For resins X and Y, however, the numbers of the viablecells were only reduced to a similar level of the control material (glass), and not reachedto 1/100 of the inoculated number. This result indicates that these resins have relativelylow antibacterial activities. Unfortunately, we could not obtain detailed information aboutthe purchased resins, but surface analysis by energy-dispersive X-ray spectroscopy (EDX)suggests the existence of the oxide of titanium and barium (Table S1). Additionally, zincwas detected on resin X, whereas a trace amount of Ag was detected on resin Y. Basedon this analysis, both resins are considered to have titanium dioxide coating, which iswell-known to have an antibacterial effect owing to its photocatalytic activity [27,28]. Inthe present study, the antibacterial assay was carried out by the film method in the safetycabinet with fluorescent light at ambient temperature, therefore, the lighting condition maynot be optimal for the photocatalysis of these resins.The antibacterial activity levels of the testing materials were indicated by the param-eters T1/10 or T1/100, as shown in Table 1. The necessary antibacterial activity level of amaterial might differ depending on its application. For example, touch surfaces in a surgicaloperation room or a hospital room require relatively high levels of antimicrobial activities,whereas those in a bathroom or a kitchen of a house for a healthy person might not requirethe same, severe levels of it. It is reasonable to suppose that the antibacterial activity levelsrequired for a touch surface depend on its purpose and usage environment. Therefore, theparameters such as T1/10 or T1/100 are useful information to choose appropriate materialsfor various touch surfaces.3.2. Nucleic Acid Degradability of Copper and Copper AlloysThough it is not fully understood, the following four mechanisms are suggested tobe involved in the process of bacterial death on copper surfaces [4,20]; (1) membraneruptures, (2) Cu2+ uptake and accumulation into the bacterial cell, (3) generation of reactiveoxygen species, and (4) DNA degradation. Dominancy or sequences among these processesremains unclear; it may be different depending on the type of microorganisms [4]. As de-scribed before, the degradation of bacterial genes is considered as beneficial to inhibit HGTof antibiotic-resistant genes and confirmed by electrophoresis of nucleic acids extractedfrom bacteria applied to the copper surface [12–18]. However, this method has a limitationon its quantitativeness since the amount of extracted nucleic acids will be influenced bythe difference in the number of viable cells recovered from the copper and the referencematerial. In order to avoid this influence, a nucleic acid solution is applied to the materialsurface and collected after a certain period of time using a swab. Since the amount of thenucleic acid solution applied is as little as 1 µL, this testing condition is closer to the actualsituation in which airborne droplets contact to the touch surface.As shown in Figure 1, the copper and its alloys have lower nucleic acid recovery rateseven at time “0” (5 min) than other materials. In the case of C1020, the recovery rate is only14.2% for dsDNA after 5 min of contact, which agrees with the result of MRSA contactingto copper, observing the disappearance of its intact DNA by staining with fluorescentintercalater [18]. The rapid decrease in the recovery rates on the copper and its alloyscorresponds well to the rapid antibacterial effect of copper and its alloys described before(Figure 9). Among the copper alloys tested, CBRI had a slightly slower decrease in therecovery rates, which agrees with its slightly larger T1/10 or T1/100. This is interestingAntibiotics 2021, 10, 1439 9 of 15since E.coli death occurs ahead of genomic DNA degradation contacting C28000 (60%Cu–40% Zn alloy) [14]. In other words, genomic DNA degradation is not considered as theprimary cause of “contact killing” on copper and its alloy surfaces, but their nucleic aciddegradability correlates to their antibacterial activity levels. It suggests that the nucleicacid degradability measured in the present study reflects the copper surface reactivity,which causes a bactericidal effect on their surfaces. Therefore, similarly to the antibacterialactivity, the nucleic acid degradability of copper and its alloys depend on the Cu content intheir composition, as described in Figure 3.Copper is known to form an oxide layer on its surface in ambient conditions byreacting with oxygen and humidity in air. Cu2O is initially formed, followed by theformation of CuO that is favored in a humid atmosphere with a long aging period [20];alloying influences the composition of the naturally formed oxide layer. For example, in thecase of copper–zinc alloys, the higher zinc content in the alloy increases ZnO in the surfaceoxide layer, suppressing Cu2O formation [29]. The difference in antibacterial activity ofCuO and Cu2O is reported; thermally prepared Cu2O layer on the pure copper surfacehas higher antibacterial activity than CuO layer [30]. Ion release from the metal surface isanother important point of view for the copper and its alloys antibacterial effect. Amongpure copper surfaces prepared by different methods as rolling, polished, and electroplated,the latter released copper ions most, resulting in the highest antibacterial activity [31].In the case of ABSS, heat treatment is necessary to enlarge the ε-Cu precipitates on thealloy surface, which encourages the release of copper ions [19,32]. The corrosion ratesof the copper alloys in 0.9% NaCl are smaller than that of pure copper, but larger thanthat of ABSS (Figure S1 and Table S2), which suggests the lower copper content in thealloy composition contributes to the decrease in copper ion release, resulting in the lowerantibacterial activity.Regarding nucleic acid degradation, the involvement of released copper ions is notclear. Therefore, nucleic acid degradation by copper salts was investigated (Figure 4).It confirmed that nucleic acid degradation can occur without copper or its alloy surfacethough their concentrations are relatively high as >1 mM for CuCl2 and >10 mM for CuCl.The copper ion release from the C1020 in the same condition to the nucleic acid degradationassay was 44–62 µM in 20 µL (Figure 5), which was over 100 times smaller than thoseof the copper salts causing the nucleic acid degradation. However, the concentrationsof copper ions released were a similar level in spite of different contact times (0–6 h)in a dry condition, suggesting that the copper ion release mainly occurred during thedrying process of the nucleic acid solution, i.e., first 5 min, or during the swabbing process(~30 s). For simplification, we suppose the copper ion release occurred during the first5 min, resulting in the copper ion concentration in the 1 µL portion of the nucleic acidsolution as 0.88–1.23 mM. The drying process reduces the amount of nucleic acid solutionto less than 1 µL, therefore, the copper ion concentration might be higher than this value.This hypothetic calculation suggests the possibility that the released copper ions may beresponsible for the nucleic acid degradation on the copper surface. In the research usingMRSA, the involvement of Cu+ and Cu2+ into genomic DNA fragmentation was confirmedby the addition of chelating reagents to the testing media [18], which coincides with theresult in the present study.Generally, the fragmentation of the bacterial gene applied to the copper surface isassigned to the generation of relative oxygen species by the Fenton-like reaction [4] shownas the following equation.Cu+ + H2O2 → ·OH + OH− + Cu2+This requires H2O2, which is not included in our experimental condition. In ournucleic acid degradation assay, nucleic acid degradation may be caused by a differentAntibiotics 2021, 10, 1439 10 of 15mechanism. In an aqueous solution, the corrosion reaction of the copper is described in thefollowing equation [33];2Cu + O2 + 2H2O→ 2Cu2+ + 4OH−where the Cu releases 2 electrons to oxygen, resulting in the generation of OH−. Thegeneration of hydroxyl ions may contribute to the fragmentation of RNA since it is easilyhydrolyzed under alkaline conditions by the nucleophilic attack from the 2-hydroxyl groupof ribose to the phosphodiester bond [34]. The affinity of divalent cations to the nucleobaseswere estimated [35] and copper ion had a relatively high affinity constant, suggesting thepossibility of chelating to the nucleobases in the single or double stranded nucleic acids.Coordination of metal ions to the nucleobases may influence the conformation of nucleicacids [35], which may enable nucleophilic attack on the phosphodiester bond from otherhydroxyl groups. Under the drying process of 1 µL of nucleic acid solution on the coppersurface, a relatively high concentration of copper ions, as well as a pH increase, may occur,which may accelerate the degradation of the nucleic acids. The higher release of copperions, that is, the higher rate of corrosion, most likely results in the higher copper ionconcentration and the higher pH, which can accelerate more the nucleic acid degradation.Further investigation is necessary to elucidate the mechanism of nucleic acid degradationon copper and its alloy surface.Recently, the degradation of viral nucleic acids on copper surface is reported [36,37],as well as denature of capsid proteins of human norovirus-like particles [38]. Thesephenomena are also considered as attributable to the high reactivity of copper and its alloysurface, which we can quantify as the nucleic acid degradability using this developedmethod. The usefulness of this method is to simulate/estimate the antibacterial/antiviralactivity of materials under the situation simulating airborne droplet contact. No biosafetyrequirement is applied to this nucleic acid degradation assay, therefore, you can performthis assay on the touch surface in the real world, such as in hospital rooms. This is one ofthe advantages of this assay and it can offer a useful measure of the antibacterial/antiviralactivity in a real environment.4. Materials and Methods4.1. A Testing MaterialsMaterials used in this study are oxygen-free copper (C1020), three kinds of copperalloys (C6932, CLEANBRIGHT® abbreviated as CBRI, and CLEANBRASS® abbreviated asCBRA), antibacterial stainless steel (NSSAM3, abbreviated as ABSS), pure silver (>99.99%),and two kinds of antibacterial resins (X and Y). All copper and copper alloys are suppliedby Mitsubishi Materials Corporation. Antibacterial stainless steel, silver, and two kinds ofresins are commercially available. Chemical compositions of oxygen-free copper and otheralloys are shown in Table 2.Table 2. Chemical compositions of oxygen free copper and alloy samples (wt.%).Sample Cu Zn Sn Ni Mn Cr Fe Si PC1020 >99.99C6932 75.4 Rem. 3.1 0.09CBRA 70.1 Rem. 0.5 2.0CBRI 54.0 Rem. 0.4 10.9ABSS 3.8 9.4 1.4 18.1 Rem. 0.6All metal samples wre cut into 15–20 mm squares and 0.5–2 mm thick for this antibac-terial assay. Specimens of antibacterial resins were cut from the purchased toilet seats andlids into 15 mm square and 3–5 mm thick. Metal specimens were ground by SiC paper upto#1200 (~5 µm), followed by rinsing with ultrapure water. Resin specimens were cleaned bywiping with absolute ethanol.Antibiotics 2021, 10, 1439 11 of 15For nucleic acid degradation assay, metal specimens of 15 cm square and 0.5–2 mmthick were prepared in the same manner except for C6932, which was prepared in disks of60 mm in diameter and 4 mm thick. For resins, ~15 mm square and 3–5 mm thick specimenswere prepared in the same manner as the antibacterial assay. The specimen surfaces werecleaned by a commercially available detergent (a mixed solution of sodium α-dodecan-1-yl-ω-(sulfonatooxy)poly(oxyethylene) and fatty acid alkanolamide) and thoroughly rinsedby ultrapure water prior to the assay.4.2. Nucleic Acid Degradation AssayThe schematic explanation of the assay procedure for nucleic acid degradation onthe material surface is shown in Figure 10. Deoxyribonucleic acid (DNA) from salmonsperm for molecular biology (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)was used as a nucleic acid source. A 1 µL portion of 20 mg/mL DNA in 0.5% poly-oxyethylene(10) octylphenyl ether (Triton X-100) solution was spread over the area about10 mm × 10 mm on the testing material surface using a tip of a digital pipette. In the caseof 15 cm samples, it was divided into four areas and one portion was loaded into each area.The surfactant was added to be easily spread the DNA solution over the sample surface.It takes ~5 min to dry the DNA portion completely. This time point is described as “0”.DNA-loaded samples were incubated at room temperature for 1, 3, and 6 h, and then, DNAportion was collected by a polyester swab with 20 µL of 0.03% sodium dodecyl sulphate(SDS). The swab was applied 20 times horizontally and 20 times vertically, alternately upto 5 sets (100 times in total) covering the DNA loaded area. The head of the swab wascut into a 480 µL portion of 0.03% SDS and vortexed for 30 sec. to extract collected DNA.Then, DNA in SDS was quantified by the acridine orange (AO) method with a calibrationcurve prepared using the dilutions of DNA solution with 0.03% SDS. A 1.2 µL portionof 0.5 mg/mL AO solution (stored at 0 ◦C) was added to a 200 µL portion of the DNAextract with SDS. AO is known to emit green (λEx = 502 nm, λEm = 526 nm) fluorescenceby binding to dsDNA whereas it emits red (λEx = 460 nm, λEm = 650 nm) fluorescence bybinding to ssDNA or RNA. Therefore, the fluorescence of the solution was measured atexcitation by a blue LED (∼470nm) and emission through a green (510–580 nm) or red(665–720 nm) filter using the benchtop fluorometer (Qubit 3.0 Fluorometer, Thermo FisherScientific KK, Tokyo, Japan) after 5 min at room temperature (22 ± 1 ◦C). The DNA extractwas diluted with 0.03% SDS, if necessary. Obtained data of AO fluorescence was convertedto the concentration of dsDNA or ssDNA + RNA based on the calibration curves preparedusing the dilutions of the DNA source. Experiments were performed in triplicate. Priorto the material evaluation, the assay condition was optimized at the following points; (1)concentrations of loading DNA and Triton X-100, (2) a type of swabbing bud (polyester,not polyurethane or cotton), (3) a swabbing protocol (no DNA was collected after 20 × 5times swabbing from glass surface loaded by DNA), and (4) concentrations of AO and SDSfor nucleic acid quantification.The nucleic acid degradability of CuCl2 and CuCl was examined in a similar mannerusing their suspension in 0.03% SDS. A 90 µL portion of 22.3 mg/mL DNA in 0.5% TritonX-100 solution was mixed with 10 µL of CuCl2 or CuCl solution appropriately diluted with0.03% SDS. After 5 min, this mixture of DNA with copper salt was diluted 100 times with0.03% SDS, and then, a 10 µL portion was mixed with 190 µL of 0.03% SDS and 1.2 µL of0.5 mg/mL AO solution. After 5 min, the fluorescence of the supernatant was measured atexcitation by a blue LED and emission through a green or red filter. If necessary, the DNAextract was diluted with 0.03% SDS. Experiments were performed in triplicate.Antibiotics 2021, 10, 1439 12 of 15Antibiotics 2021, 10, x FOR PEER REVIEW 12 of 15  dium dodecyl sulphate (SDS). The swab was applied 20 times horizontally and 20 times vertically, alternately up to 5 sets (100 times in total) covering the DNA loaded area. The head of the swab was cut into a 480 µL portion of 0.03% SDS and vortexed for 30 sec. to extract collected DNA. Then, DNA in SDS was quantified by the acridine orange (AO) method with a calibration curve prepared using the dilutions of DNA solution with 0.03% SDS. A 1.2 µL portion of 0.5 mg/mL AO solution (stored at 0 °C) was added to a 200 µL portion of the DNA extract with SDS. AO is known to emit green (λEx = 502 nm, λEm = 526 nm) fluorescence by binding to dsDNA whereas it emits red (λEx = 460 nm, λEm = 650 nm) fluorescence by binding to ssDNA or RNA. Therefore, the fluorescence of the solution was measured at excitation by a blue LED (∼470nm) and emission through a green (510–580 nm) or red (665–720 nm) filter using the benchtop fluorometer (Qubit 3.0 Fluorometer, Thermo Fisher Scientific KK, Tokyo, Japan) after 5 min at room tempera-ture (22 ± 1 °C). The DNA extract was diluted with 0.03% SDS, if necessary. Obtained data of AO fluorescence was converted to the concentration of dsDNA or ssDNA + RNA based on the calibration curves prepared using the dilutions of the DNA source. Exper-iments were performed in triplicate. Prior to the material evaluation, the assay condition was optimized at the following points; (1) concentrations of loading DNA and Triton X-100, (2) a type of swabbing bud (polyester, not polyurethane or cotton), (3) a swabbing protocol (no DNA was collected after 20 × 5 times swabbing from glass surface loaded by DNA), and (4) concentrations of AO and SDS for nucleic acid quantification.  Figure 10. Schematic explanation of the procedure for the evaluation of the nucleic acid degrada-bility of material surfaces by a swab method. The nucleic acid degradability of CuCl2 and CuCl was examined in a similar man-ner using their suspension in 0.03% SDS. A 90 µL portion of 22.3 mg/mL DNA in 0.5% Triton X-100 solution was mixed with 10 µL of CuCl2 or CuCl solution appropriately di-luted with 0.03% SDS. After 5 min, this mixture of DNA with copper salt was diluted 100 times with 0.03% SDS, and then, a 10 µL portion was mixed with 190 µL of 0.03% SDS and 1.2 µL of 0.5 mg/mL AO solution. After 5 min, the fluorescence of the superna-tant was measured at excitation by a blue LED and emission through a green or red fil-ter. If necessary, the DNA extract was diluted with 0.03% SDS. Experiments were per-formed in triplicate. 4.3. Measurement of Copper Ion Release Copper ion release from C1020 during the nucleic acid degradation assay was measured by the following procedure. A 1 µL portion of 0.5% Triton X-100 was spread in the same manner to the nucleic acid degradation assay on the C1020 specimen sur-face. It takes ~5 min to dry completely, which is the time point of “0”. The specimens Figure 10. Schematic explanation of the procedure for the evaluation of the nucleic acid degradabilityof material surfaces by a swab method.4.3. Measurement of Copper Ion ReleaseCopper ion release from C1020 during the nucleic acid degradation assay was mea-sured by the following procedure. A 1 µL portion of 0.5% Triton X-100 was spread in thesame manner to the nucleic acid degradation assay on the C1020 specimen surface. It takes~5 min to dry completely, which is the time point of “0”. The specimens were incubated atroom temperature for 1, 3, and 6 h, and then its surface was swabbed in the same manner asthe nucleic acid degradation assay. The head part of the swab was cut into a 475 µL portionof 0.03% SDS and mixed (shaken) for 30 sec. to extract copper ions. Then, copper ions inthis extract were quantified using the Metallo Assay Copper Low Concentrate Assay KitLS (Metallogenics Co. Ltd., Chiba, Japan) following its instruction supplied with the kit.Briefly, a 396 µL portion of the extract was added to 4 µL 1 M hydrochloric acid (HCl) toadjust its pH as 2–3. Then, a 100 µL portion was added to 140 µL of the mixture of thebuffer and chelate color reagent. After 10 min at room temperature, absorbance at 590 nmwas measured by a microplate reader (Multiskan FC, Thermo Fischer Scientific KK, Tokyo,Japan). Obtained absorbance was converted to the concentration of copper, based on acalibration curve prepared using the dilutions of copper standard solution. Experimentswere performed in triplicate.4.4. Antibacterial AssayThe bacterial cell lines used were Escherichia coli (ATCC 8739, EZ-PECTM, Microbiologics®,purchased from EnBio Ltd., Tokyo, Japan) and Staphylococcus aureus (ATCC 6538, EZ-PECTM, Microbiologics®, purchased from EnBio Ltd., Tokyo, Japan). The former wasemployed as a representative of Gram-negative bacteria, whereas the latter was a Gram-positive. Each of the testing materials was placed on the bottom of the glass dish separately.A 50 µL portion of cell suspension containing ~1 × 106 cells in 0.9% NaCl were placedonto a testing material and covered by a polyethylene film of 10 mm square to control thecontacting area of the bacterial suspension. As a control surface, bacterial cells were loadedto the bottom of a glass dish in the same manner. Then, the samples were incubated at35 ± 1 ◦C and relative humidity over 90% for 5, 10, 30, 60, 120, or 1440 min. After incuba-tion, 1 mL of the mixture of 0.9% NaCl and 0.1 mM ethylenediamine-N,N,N’,N’-tetraaceticacid, disodium salt, dihydrate (EDTA-2 Na) was poured and pipetted three times over thesample in the dish to collect survived bacterial cells. Then, a 200 µL portion was used fora viable cell count using 5-cyano-2,3-ditolyl-2H-tetrazolium chloride (CTC) staining kit(-Bacstain- CTC Rapid Staining Kit for Flow cytometry, BS01, Dojindo Laboratories, Ku-mamoto, Japan) following its instruction. Briefly, 4.2 µL of the mixture of CTC and enhancerAntibiotics 2021, 10, 1439 13 of 15reagent was added. After incubation for 30 min at 35 ± 1 ◦C, fluorescence was measuredat excitation through blue (430–495 nm) filter and emission through red (665–720 nm) filterusing a benchtop fluorometer. Obtained data of CTC fluorescence was converted to thenumber of cells based on the calibration curves prepared using the dilutions of bacterialcell suspension. All the experiments were performed at least in duplicate and in triplicatewhere necessary.5. ConclusionsIn the present study, nucleic acid degradability of Cu and its alloy surfaces wasquantitatively evaluated by a new in vitro method and compared to those of commerciallyavailable antibacterial resins, antibacterial stainless steel, and pure Ag. As a result, onlyCu and its alloys showed continuous degradation of nucleic acids up to 6h of contacttime, which may be attributed to the copper ion release from the Cu and its alloy surfaces.The nucleic acid degradability evaluated in this new method has a good correlation tothe antibacterial levels evaluated by a film method. The higher Cu content in the alloycomposition results in the higher nucleic acid degradability and the higher antibacterialactivities.Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/antibiotics10121439/s1, Figure S1: Potentiodynamic curves of copper and copper-containingalloys in 0.9% NaCl, Table S1: Results of surface analysis of commercially available antibacterialresins (wt%), and Table S2: Corrosion parameters of copper and its alloys in 0.9% NaCl.Author Contributions: Conceptualization, A.Y., S.T., and K.O.; methodology, A.Y.; formal analysis,A.Y.; investigation, A.Y.; resources, A.Y., S.T., K.O.; data curation, A.Y.; writing—original draftpreparation, A.Y.; writing—review and editing, A.Y., S.T., and K.O.; visualization, A.Y.; supervision,A.Y. and K.O.; project administration, A.Y. and S.T.; funding acquisition A.Y. and K.O. All authorshave read and agreed to the published version of the manuscript.Funding: This research received no external funding.Data Availability Statement: The data presented in this study are available on request from thecorresponding author.Acknowledgments: A.Y. would like to thank Yuko Kohyama and Akemi Kikuta for their technicalassistance with the antibacterial assay and the potentiodynamic measurement. The authors appreciateKomei Kato and Yasuharu Hakamatsuka for their advice on the discussion of obtained data and theproject administration.Conflicts of Interest: S.T. and K.O. are employed by Mitsubishi Materials Corporation. The fundershad no role in the design of the study; in the collection, analyses, or interpretation of data; in thewriting of the manuscript, or in the decision to publish the results.References1. Review on Antimicrobial Resistance. 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[CrossRef] [PubMed]http://doi.org/10.1128/mBio.01697-15http://www.ncbi.nlm.nih.gov/pubmed/26556276http://doi.org/10.1128/AEM.00388-15http://www.ncbi.nlm.nih.gov/pubmed/25979897 Introduction  Results  Nucleic Acid Degradability of Testing Materials  Nucleic Acid Degradation by Copper Salts  Antibacterial Activity of Testing Materials  Correlation between Nucleic Acid Degradability and Antibacterial Activity of Testing Materials  Discussion  Antibacterial Activity of Copper, Copper Alloys, and Other Antibacterial Materials  Nucleic Acid Degradability of Copper and Copper Alloys  Materials and Methods  A Testing Materials  Nucleic Acid Degradation Assay  Measurement of Copper Ion Release  Antibacterial Assay  Conclusions  References