Inhibition of Staphylococcus aureus biofilm by a copper-bearing 317L-Cu stainless steel and its corrosion resistance

Materials Science and Engineering C 69 (2016) 744–750

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Inhibition of Staphylococcus aureus biofilm by a copper-bearing 317L-Cu stainless steel and its corrosion resistance Da Sun a,1, Dake Xu b,⁎,1, Chunguang Yang b, Jia Chen a, M. Babar Shahzad b, Ziqing Sun b, Jinlong Zhao b, Tingyue Gu c, Ke Yang b, Guixue Wang a,⁎ a Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400030, China b Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang 110016, China c Department of Chemical and Biomolecular Engineering, Institute for Corrosion and Multiphase Technology, Ohio University, Athens, OH 45701, USA

a r t i c l e

i n f o

Article history: Received 25 April 2016 Received in revised form 12 July 2016 Accepted 19 July 2016 Available online 20 July 2016 Keywords: 317L-Cu Antibacterial Staphylococcus aureus Biofilm Cytotoxicity

a b s t r a c t The present study investigated the antibacterial performance, corrosion resistance and surface properties of antibacterial austenitic 317L-Cu stainless steel (317L-Cu SS). After 4.5 wt% copper was added to 317L stainless steel (317L SS), the new alloy underwent solid solution and aging heat treatment. Fluorescent staining using 4′,6diamidino-2-phenylindole (DAPI) revealed that the 317L-Cu SS showed strong antibacterial efficacy, achieving a 99% inhibition rate of sessile Staphylococcus aureus cells after 5 days. The corrosion data obtained by potentiodynamic polarization curves indicated that in comparison with 317L SS, the pitting potential and corrosion current density of 317L-Cu slightly decreased due to the addition of Cu. The 317L-Cu SS exhibited no cytotoxicity against zebrafish (Danio rerio) embryos. The experimental results in this study demonstrated that the new alloy has potential applications in medical and daily uses. © 2016 Elsevier B.V. All rights reserved.

1. Introduction With the advances of biomaterials for biomedical applications, biomaterial-related infections are becoming more prevalent than ever before [1]. Biofilm is the main cause of biomedical implant related infections with serious consequences such as implant failures, systemic and repeated bacterial infections [2,3]. In spite of an aseptic operation, the infection rate after total hip arthroplasty was 0.5–3.0%, but it could reach 5.0–35.0% in prosthesis replacement [4,5]. More than half of orthopaedic implant-related infections are caused by staphylococci spp., wherein Staphylococcus aureus contributes 51.5% [6]. Moreover, the Gram-positive S. aureus is a common cause of infection on implanted catheters and other medical devices, and is notoriously resistant to antibiotics [7–9]. Using an antimicrobial material is one of the most effective approaches to mitigate persistent biofilm related infections. The surface modification technology is becoming a hot research area in recent years. A major problem of this approach is the unstable bonding between coatings and metal surfaces, and the unsustainable release of antibacterial agents. These drawbacks hamper their practical applications [10,11]. Stainless steel (SS) is one of the most common materials used in ⁎ Corresponding authors. E-mail addresses: [email protected] (D. Xu), [email protected] (G. Wang). 1 These two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.msec.2016.07.050 0928-4931/© 2016 Elsevier B.V. All rights reserved.

the biomedical field. 316L SS which contain 2.0–3.0% molybdenum are primarily applied in various implantable and non-implantable medical devices due to its good balance of mechanical properties, corrosion resistance, biocompatibility and cost. 317L SS containing 3.0–4.0% molybdenum can be used in applications where enhanced general corrosion and pitting corrosion resistance are required [12]. The applications of SS in the clinical field may extensively expand if it possesses an antibacterial ability. Nisshin Steel (Tokyo, Japan) first developed antibacterial copper-containing SS in the 1990s [13]. Since the beginning of this century, Institute of Metal Research (IMR), Chinese Academy of Sciences (Shenyang, China) has developed various types of copper bearing SS including ferrite, austenite and martensite [14–16]. Cu is one of the most essential trace elements present in human body, and it also exhibits strong antibacterial performance [13,17–20]. The broad-spectrum antimicrobial ability of antibacterial SS is mainly due to the copper ions released from its surface, which can damage the bacterial cell walls and cell membranes, adsorb electrons from bacteria, generate the reactive oxygen species (ROS), resulting in the fatal damage and death of the bacteria [21–23]. Apart from the excellent sustained antibacterial ability, these antimicrobial copper-bearing SS possessed strong resistance to high temperature with good plasticity [24,25]. Therefore, copper-bearing SS as an antibacterial material has great potentials in healthcare and food industry applications. In this study, 4′,6-diamidino-2-phenylindole (DAPI) staining was used to evaluate the antibacterial performance of the 317L-Cu SS. Its

D. Sun et al. / Materials Science and Engineering C 69 (2016) 744–750

hardness, contact angle, and corrosion resistance were also measured. Furthermore, the extracellular polymeric substances (EPS) formed on 317L SS (control) and the 317L-Cu SS were analyzed using Fourier transform infrared spectrum (FTIR). Finally, the in vivo toxicity of the 317L-Cu SS was investigated using zebrafish embryos. 2. Materials and methods 2.1. Materials The chemical compositions of 317L and 317L-Cu are listed in Table 1, both provided by IMR. A 317L-Cu specimen was first annealed at 1100 °C for 0.5 h and quenched in water, then aged at 700 °C for 6 h in order to precipitate the Cu-rich phases in the alloys [26]. The heat-treated specimen and an untreated 317-L specimen (control) were cut into coinshape coupons with 10 mm in diameter and 1.5 mm thickness. The coupons were abraded sequentially with a series of SiC papers (150, 400, 600, 800, 1000 and 1200 grit). The coupons were cleaned using acetone prior to the experiments [27]. All the coupons were dried in a superclean bench under UV light before use. 2.2. Biofilm mitigation test S. aureus (ATCC 25923) was obtained from Guangzhou Institute of Microbiology (Guangdong, China). The 317L and 317L-Cu coupons were placed in a 24-multiwell culture plate. The S. aureus suspension of 1 ml at the concentration of 106 CFU ml− 1 was added into each well containing a coupon. The 24-multiwell culture plates with S. aureus and coupons were incubated in a DNP 9272 electric thermostatic incubator (Jinghong Co., Shanghai, China) at 37 °C for 0.5, 1, 3 and 5 days, respectively. Quintuplicate coupons were prepared for each time point. To evaluate the antibacterial performance of 317L-Cu, fluorescent staining with DAPI (Sigma-Aldrich Co., MO, USA) was used. After incubation for 0.5, 1, 3 and 5 days, 1 μl of 5 m mol l−1 DAPI was added to each well. Coupons were then incubated for 0.5 h at room temperature in the dark. After being gently cleaned using 0.9% (w/v) NaCl followed by a pH 7.4 PBS buffer solution, the coupons were thoroughly examined using confocal laser scanning microscopy (CLSM, Model C2si+; Nikon Co., Kyoto, Japan). For each coupon examined, representative images were taken using Nis-Elements Viewer version 4.20 (Nikon Co., Kyoto, Japan) [28]. The images of the biofilms on the 317L and 317L-Cu coupons were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA) to count the sessile cells [29]. 2.3. EPS and biofilm analysis FTIR was employed to analyze the EPS of the S. aureus biofilms on the 317L and 317-L Cu coupons. The coupons were incubated with S. aureus in 0.9% (w/v) NaCl solution at the initial cell concentration of 106 CFU ml−1 for 5 days to ensure the coverage of the biofilm on the coupon surface. The coupons were then dried through an infrared light bulb in a SW-CJ-1FD super-clean bench (Antai Airtech Co., Ltd., Suzhou, China). A FTIR spectrometer was employed to characterize infrared signatures scraped from dried surfaces [10]. The coupon surfaces after incubation with S. aureus were analyzed using an ESCALAB250 X-ray photoelectron spectroscopy (XPS, Thermo VG, Waltham, USA). XPS measurements were performed with monochromatic Al kα radiation at 15 kV and 150 W. The pass energy of the Table 1 Chemical compositions of 317L SS and 317L-Cu SS (wt%). Element

Ni

Cr

Mo

Cu

Fe

317L-Cu 317L

15.15 14.55

18.25 19.22

3.72 3.64

4.46 –

Balance Balance

745

spectra ranged within 0–1350 eV, and the passing energy to record the high resolution spectra was 50 eV with 0.1 eV step. 2.4. Surface characterization The chemical compositions (wt%) of the surfaces were characterized using a scanning electron microscope (SEM, Ultra Plus, Jena, Germany) with energy dispersive spectrometer (EDS). The hardness of the coupons was measured using a HVT-1000 micro-hardness tester (Beilun Jiankun Control Equipment Co., Ningbo, China) with a Vickers indenter at a load of 0.245 N. The contact angle of the coupons was measured with distilled water and a SZ10-JC2000A contact angle device (Zhongchen Digital Technic Apparatus Co., Shanghai, China) [30]. The surface free energy (SFE) was calculated using the Owen's method, and the test liquids used in this study were deionized water and 1bromonaphthalene [16,31–34]. 2.5. Copper ion concentration The 317L-Cu coupons were immersed in 5 ml centrifuge tubes containing 2 ml of the 0.9% NaCl solution. The Cu2+ ion concentration released from 317L-Cu SS into the 0.9% NaCl solution after 0.5, 1, 3, 5 days was measured using an atomic absorption spectrophotometer (Model Z-2000; Hitachi Ltd., Tokyo, Japan) following the procedures reported in a previous study [35]. 2.6. Electrochemical test The corrosion resistance of the 317L and 317L-Cu was evaluated with potentiodynamic polarization curves [35]. A simulated body fluid solution (0.9% NaCl) was used as the electrolyte for electrochemical tests, and a potentiostat–galvanostat (Reference 600™, Gamry Instruments, Inc., Warminster, USA) electrochemical workstation was employed to evaluate the electrochemical behaviors of the coupons. The electrolytes were kept at 37 ± 0.5 °C, and a saturated calomel electrode (SCE) was used as the reference electrode. The potentiodynamic polarization curves of each coupon were measured at a scan rate of 0.5 mV s− 1 after the open circuit potential (OCP) was measured. To check reproducibility, each experiment was conducted three times. 2.7. In vivo toxicity study The Chinese Animal Care and Use Committee Standards were followed for the animal housing and surgical procedures. All procedures were done in accordance with protocols approved by the Animal Ethics Committee of Chongqing University. Ten healthy zebrafish embryos (eight-cell stage) were transferred to each well of 24-well plates [36]. The embryos after incubation with 317L-Cu SS coupons were observed at specific growth stages for hatching rates, growth, abnormality and survival rates, and touch responses of larval zebrafish [37]. At the end of the experiment, the larval zebrafish were transferred to a microscope slide and then anesthetized with 0.03% (w/v) MS-222 (Sigma Co., MO, USA). Microscopic observations were performed using an Olympus SZX10 microscope (Olympus Co., Tokyo, Japan) to investigate the development of the embryos and larval zebrafish. 3. Results and discussion 3.1. Antibacterial performance Because of the viable but non-cultural (VBNC) state of S. aureus, the plate-count method cannot fully reveal the antibacterial performance of the 317L-Cu [38]. The DAPI staining method was used to evaluate its biofilm mitigation ability instead [39]. Fig. 1(a1–a4, b1–b4) presents the comparison of the biofilms after DAPI staining on the 317L and 317L-Cu surfaces after 0.5, 1, 3, 5 days, indicating the strong inhibition

746

D. Sun et al. / Materials Science and Engineering C 69 (2016) 744–750

Fig. 1. CLSM images of sessile S. aureus cells on 317L SS after 0.5 (a1), 1 (a2), 3 (a3) and 5 days (a4) of incubation, and on 317L-Cu SS after 0.5 (b1), 1 (b2), 3 (b3) and 5 days (b4) of incubation, and the time-changing trend diagram with number of bacterial colonies (c). (Scale bar = 50 μm).

D. Sun et al. / Materials Science and Engineering C 69 (2016) 744–750

a

Cu2p

Mean ± standard deviation (n = 10).

Cl2p

16.2 ± 0.7

C1s

5

9.5 ± 0.49

O1s

3

5.3 ± 0.33

Cr2p

1

2.2 ± 0.12

Fe2p

0.5

317L SS 317L-Cu SS

Na1s

Time (day) Concentration of Cu2+ (ppb)a

Mg1s

Table 2 Cu2+ released from 317L-Cu in 0.9% NaCl over time.

a

747

1150-1030 1649

1400

1200

1000

800

600

400

200

0

Binding energy, Eb / eV

1428 77000

b

1150-1030

Fig. 2. FTIR spectra of EPS on 317L SS and 317L-Cu SS surfaces.

effect of the 317L-Cu. The numbers of sessile bacterial colonies were significantly reduced (1.4 order of magnitude) on the 317L-Cu surface after 5 days as shown in Fig. 1c. The antibacterial efficacy of the 317L-Cu increased with longer exposure time, demonstrating that continuous copper ions released enhanced its biocidal effect. Meanwhile there was an increased concentration of Cu2+ released from the 317L-Cu over time as shown in Table 2, explaining its excellent antibacterial performance [40]. The Cu2+ was continuously released from the 317L-Cu, resulting in the strong inhibition of the S. aureus biofilm. The Cu2+ concentration after 5 days incubation reached 16.2 ± 0.7 ppb, the local concentration of Cu2+ underneath the biofilm could be far more than that in the bulk solution. 3.2. The investigation of EPS and biofilm To form a biofilm, EPS is secreted by bacteria [41]. As can be seen from Fig. 2 for the 317L and 317L-Cu coupons after 5 day incubation,

Element

Weight%

Atomic%

Cr

18.09

19.74

Fe

58.19

59.05

Ni

15.59

15.07

Cu

4.38

3.92

Mo

3.75

2.22

Intensity (a.u.)

1428

3500-3100

317L-Cu SS

76000

75000

Cu CuO

74000

73000

72000 936

934

932

930

928

Binding energy, Eb / eV Fig. 4. (a) Wide XPS spectra of the surfaces of 317L SS and 317L-Cu SS incubated with S. aureus for 5 days, and (b) high resolution XPS spectra of Cu 2p for 317L-Cu SS after incubation with S. aureus for 5 days.

there were stretching vibrations of N\\H and O\\H (3500– 3100 cm− 1), C_O (1648.5 cm− 1), CH2 (1428.2 cm−1), the C\\O\\C of polysaccharide (1150–1030 cm− 1) and the fingerprint spectrum (b1000 cm−1). These data indicated that 317L-Cu strongly influenced the EPS composition, the peaks of N\\H, O\\H and C_O were

Fig. 3. SEM image of 317L-Cu bare coupon surface and its corresponding EDS analysis.

748

D. Sun et al. / Materials Science and Engineering C 69 (2016) 744–750

eliminated which illustrated that these groups were involved in the coordination reaction [42]. The hindrance of EPS formation could lead to the inhibition of the biofilm formation [43].

0.6

317L SS 317L-Cu SS

0.4

Fig. 3 shows the SEM image of a 317L-Cu bare coupon surface without incubation with S. aureus, and the corresponding EDS analysis data. The surface composition was analyzed using XPS. Fig. 4a shows the wide spectra of the layer. The Cu 2p spectra obtained from the 317L-Cu SS coupons after incubation with S. aureus for 5 days are shown in Fig. 4b, which exhibited a stronger peak at the binding energy of ~932.7 eV corresponding to the species of Cu and CuO [44], indicating that the biocidal function of 317L-Cu SS may attribute to the copper ions released from the ɛ-Cu phases in its matrix [13,45]. Nevertheless, the hydrophobicity and SFE of the material surface can also affect the bacterial growth and propagation significantly [11,46]. Fig. 5 indicates that there were no significant differences of the contact angle values between the 317L and 317L-Cu coupons, and both of them were always hydrophilic after 5 days, which benefits the biocompatibility of the materials [47]. Besides, the contact angles of 317L SS decreased while the SFE of 317L SS increased after being incubated with S. aureus. The nonpolar components of both SS remained a stable range from 35 to 38 mJ m−2. The polar component and SFE of 317L were always slightly

a

b

317L SS 317L-Cu SS

317L SS 317L-Cu SS

E (V vs SCE)

3.3. Surface properties 0.2

0.0

-0.2

-0.4

-0.6 -10

-9

-8

-7

-6

-5

-4

-3

-2

Log i (A cm ) Fig. 6. Potentiodynamic polarization curves of 317L SS and 317L-Cu SS after 5 day incubation in a 0.9% NaCl solution.

higher than those of 317L-Cu, which could attribute to the organic compounds produced by S. aureus, compositions of the alloys and the heat treatment process [46,48,49]. The SFE of 317L-Cu decreased at 5 days, this might be caused by the less adhesion of S. aureus biofilm as shown in Figures 1 a4 and b4, resulting in a decrease of SFE [50]. The results of hardness test show that, compared with the 317L SS (160.2 ± 1.2 kgf mm−2), the hardness of the 317L-Cu (166.8 ± 3 kgf mm− 2) was much closer to the commercially pure titanium (166.3– 181.9 kgf mm−2). Fig. 6 shows the potentiodynamic polarization curves of 317L and 317L-Cu coupons in a 0.9% NaCl solution after 5 day incubation. The pitting potential of 317L and 317L-Cu were 508.5 mV and 463.5 mV, respectively (Table 3). The heat treatment processes were intended to precipitate the ɛ-Cu phases in the austenitic SS matrix. Thus it was possible that the ɛ-Cu phases acted as a cathodic center, thereby accelerating the dissolution of the matrix which can affect the corrosion property of 317L-Cu [51]. The slightly decreased pitting potential of 317L-Cu was consistent with the results reported in a reference [33]. The copper addition might also influence the structure of the passivation film, which was the most important factor to influence the corrosion resistance of the 317L-Cu SS. Its icorr was 136.7 nA cm−2, which was larger than that of 317L (113.8 nA cm− 2), indicating that the addition of copper did not significantly decrease its corrosion resistance under this mild corrosion environment.

Table 3 The polarization parameters of 317L SS and 317L-Cu SS in the 0.9% NaCl solution after 5 day incubation. Material

Ecorr/mV

βa/mV dec−1

βc/mV dec−1

icorr/nA cm−1

Ep/mV

317L SS 317L-Cu SS

−133.9 −176.2

0.44 0.32

0.197 0.094

113.8 136.7

508.5 463.5

Table 4 Toxic effects of 317L-Cu SS against zebrafish embryos. Control (317L)

Fig. 5. (a) The contact angles, and (b) the SFE of the SS coupons after incubation with S. aureus at 37 °C after 0.5, 1, 3, 5 days.

317L-Cu SS

Stages of embryos

48 h (%)

72 h (%)

48 h (%)

72 h (%)

Hatching rate of embryos Abnormal rate of embryos Abnormal rate of larval zebrafish Mortality

100 0 – 0

– – 0 0

100 0 – 0

– – 0 0

D. Sun et al. / Materials Science and Engineering C 69 (2016) 744–750

749

a2

a1

317L SS b1

317L SS b2

317L-Cu SS

317L-Cu SS

Fig. 7. In vivo toxicity analysis of the 317L-Cu SS against zebrafish embryos: unhatched embryos at 48 h (a1, b1), hatched embryos at 72 h (a2, b2).

3.4. In vivo cytotoxicity evaluation Zebrafish shares some similar biological structures and physiological functions with mammals and it has been widely used as a simple biological test system [52]. This animal model was used in toxicity studies due to its large sample volume, low cost, and short test cycle [53]. The data in Table 4 suggest that the rate and time of hatching, mortality and touch response of the larval zebrafish were similar in the 317L SS control samples and in the 317L-Cu SS-incubated samples. There were no malformations on embryos and larval zebrafish under microscopic observations (Fig. 7). Our results demonstrated that the early development of zebrafish were unaffected by the 317L-Cu SS, suggesting its excellent biocompatibility with animals.

4. Conclusions This study clearly demonstrated that the 317L-Cu SS with special heat treatment processes possessed excellent antibacterial activity against the S. aureus biofilm. In addition, the results of in vitro cytotoxicity studies suggested that the 317L-Cu SS had good biocompatibility with animals. It also had better corrosion resistance than the 317L SS control. Therefore, the 317L-Cu SS may have many potential applications in surgical instruments, orthopaedic furniture and outside surface of clinical equipment with an anti-infective function.

Acknowledgments This study was funded by grants from the National Natural Science Foundation of China (11332003, 31370949, 81400329), the National Key Research and Development Program (2016YFC1102305), the “Young Merit Scholars” program of the Institute of Metal Research, Chinese Academy of Sciences, the Chongqing Engineering Laboratory in

Vascular Implants, the National “111 Plan” Base (B06023) and the Public Experiment Center of the State Bioindustrial Base (Chongqing) of China.

References [1] C.R. Arciola, D. Campoccia, P. Speziale, L. Montanaro, J.W. Costerton, Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials, Biomaterials 33 (2012) 5967–5982, http://dx.doi.org/10.1016/j.biomaterials.2012.05.031. [2] S. Zhang, C. Yang, G. Ren, L. Ren, Study on behaviour and mechanism of Cu2+ ion release from Cu bearing antibacterial stainless steel, Mater. Technol. (2014) http://dx. doi.org/10.1179/1753555714Y.0000000236. [3] J.W. Costerton, P.S. Stewart, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections, Science 284 (1999) 1318–1322, http://dx.doi.org/10.1126/ science.284.5418.1318. [4] P. Anguita-Alonso, A.D. Hanssen, R. Patel, Prosthetic joint infection, Expert Rev. AntiInfect. Ther. 3 (2005) 797–804, http://dx.doi.org/10.1586/14787210.3.5.797. [5] X. Chen, X. Li, Extracorporeal shock wave therapy could be a potential adjuvant treatment for orthopaedic implant-associated infections, J. Med. Hypotheses Ideas 7 (2013) 54–58, http://dx.doi.org/10.1016/j.jmhi.2013.03.002. [6] D. Campoccia, L. Montanaro, C.R. Arciola, The significance of infection related to orthopedic devices and issues of antibiotic resistance, Biomaterials 27 (2006) 2331–2339, http://dx.doi.org/10.1016/j.biomaterials.2005.11.044. [7] M.C. Brouwer, A.R. Tunkel, D. van de Beek, Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis, Clin. Microbiol. Rev. 23 (2010) 467–492, http://dx.doi.org/10.1128/CMR.00070-09. [8] L.G. Miller, F. Perdreau-Remington, G. Rieg, S. Mehdi, J. Perlroth, A.S. Bayer, A.W. Tang, T.O. Phung, B. Spellberg, Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles, N. Engl. J. Med. 352 (2005) 1445–1453, http://dx.doi.org/10.1056/NEJMoa042683. [9] R.P. Schuenck, M.C.S. Lourenco, N.L.P. Iório, A.L.P. Ferreira, S.A. Nouér, K.R.N. Santos, Improved and rapid detection of methicillin-resistant Staphylococcus aureus nasal carriage using selective broth and multiplex PCR, Res. Microbiol. 157 (2006) 971–975, http://dx.doi.org/10.1016/j.resmic.2006.08.004. [10] J. Liu, D. Zhang, X. Pan, L. Wang, Characterization of the complexation between Al3+ and extracellular polymeric substances prepared from alga-bacteria biofilm, Chin. J. Appl. Environ. Biol. 15 (2009) 347–350, http://dx.doi.org/10.3724/SP.J.1145.2009. 00347. [11] W. Zhao, S.L. Walker, Q. Huang, P. Cai, Contrasting effects of extracellular polymeric substances on the surface characteristics of bacterial pathogens and cell attachment to soil particles, Chem. Geol. 410 (2015) 79–88, http://dx.doi.org/10.1016/j. chemgeo.2015.06.013.

750

D. Sun et al. / Materials Science and Engineering C 69 (2016) 744–750

[12] Q. Chen, G.A. Thouas, Metallic implant biomaterials, Mater. Sci. Eng. R. Rep. 87 (2015) 1–57, http://dx.doi.org/10.1016/j.mser.2014.10.001. [13] D. Sun, M. Babar Shahzad, M. Li, G. Wang, D. Xu, Antimicrobial materials with medical applications, Mater. Technol. 30 (2014) B90–B95, http://dx.doi.org/10.1179/ 1753555714Y.0000000239. [14] L. Nan, Y. Liu, M. Lue, K. Yang, Study on antibacterial mechanism of copper-bearing austenitic antibacterial stainless steel by atomic force microscopy, J. Mater. Sci. Mater. Med. 19 (2008) 3057–3062, http://dx.doi.org/10.1007/s10856-008-3444-z. [15] D. Zhang, L. Ren, Y. Zhang, N. Xue, K. Yang, M. Zhong, Antibacterial activity against Porphyromonas gingivalis and biological characteristics of antibacterial stainless steel, Colloids Surf. B: Biointerfaces 105 (2013) 51–57, http://dx.doi.org/10.1016/ j.colsurfb.2012.12.025. [16] M. Li, L. Nan, D. Xu, G. Ren, K. Yang, Antibacterial performance of a Cu-bearing stainless steel against microorganisms in tap water, J. Mater. Sci. Technol. 31 (2015) 243–251, http://dx.doi.org/10.1016/j.jmst.2014.11.016. [17] T.N. Kim, Q.L. Feng, J.O. Kim, J. Wu, H. Wang, G.C. Chen, F.Z. Cui, Antimicrobial effects of metal ions (Ag+, Cu2+, Zn2+) in hydroxyapatite, J. Mater. Sci. Mater. Med. 9 (1998) 129–134. [18] A. Golcu, M. Tumer, H. Demirelli, R.A. Wheatley, Cd(II) and Cu(II) complexes of polydentate Schiff base ligands: synthesis, characterization, properties and biological activity, Inorg. Chim. Acta 358 (2005) 1785–1797, http://dx.doi.org/10.1016/j. ica.2004.11.026. [19] K. Singh, M. Barwa, P. Tyagi, Synthesis, characterization and biological studies of Co(II), Ni(II), Cu(II) and Zn(II) complexes with bidentate Schiff bases derived by heterocyclic ketone, Eur. J. Med. Chem. 41 (2006) 147–153, http://dx.doi.org/10. 1016/j.ejmech.2005.06.006. [20] J.P. Ruparelia, A.K. Chatterjee, S.P. Duttagupta, S. Mukherji, Strain specificity in antimicrobial activity of silver and copper nanoparticles, Acta Biomater. 4 (2008) 707–716, http://dx.doi.org/10.1016/j.actbio.2007.11.006. [21] C.E. Santo, E.W. Lam, C.G. Elowsky, D. Quaranta, D.W. Domaille, C.J. Chang, G. Grass, Bacterial killing by dry metallic copper surfaces, Appl. Environ. Microbiol. 77 (2011) 794–802, http://dx.doi.org/10.1128/AEM.01599-10. [22] S.L. Warnes, C.W. Keevil, Mechanism of copper surface toxicity in vancomycin-resistant Enterococci following wet or dry surface contact, Appl. Environ. Microbiol. 77 (2011) 6049–6059, http://dx.doi.org/10.1128/AEM.00597-11. [23] S.L. Warnes, V. Caves, C.W. Keevil, Mechanism of copper surface toxicity in Escherichia coli O157:H7 and Salmonella involves immediate membrane depolarization followed by slower rate of DNA destruction which differs from that observed for Gram-positive bacteria, Environ. Microbiol. 14 (2012) 1730–1743, http://dx. doi.org/10.1111/j.1462-2920.2011.02677.x. [24] F. Shi, X. Li, Y. Hu, C. Su, C. Liu, Optimization of grain boundary character distribution in Fe-18Cr-18Mn-0.63N high-nitrogen austenitic stainless steel, Acta Metall. Sin. Engl. Lett. 26 (2013) 497–502, http://dx.doi.org/10.1007/s40195-013-0323-5. [25] T. Ma, Y. Li, Development situation and application prospect of antibacterial stainless steels, Mater. Rev. (2015) 98–101,127, http://dx.doi.org/10.11896/j.issn.1005023X.2015.013.018. [26] L. Ren, J. Zhu, L. Nan, K. Yang, Differential scanning calorimetry analysis on Cu precipitation in a high Cu austenitic stainless steel, Mater. Des. 32 (2011) 3980–3985, http://dx.doi.org/10.1016/j.matdes.2011.03.068. [27] J.O. Noyce, H. Michels, C.W. Keevil, Potential use of copper surfaces to reduce survival of epidemic meticillin-resistant Staphylococcus aureus in the healthcare environment, J. Hosp. Infect. 63 (2006) 289–297, http://dx.doi.org/10.1016/j.jhin.2005.12. 008. [28] L. Weaver, J.O. Noyce, H.T. Michels, C.W. Keevil, Potential action of copper surfaces on meticillin-resistant Staphylococcus aureus: action of copper surfaces on MRSA, J. Appl. Microbiol. 109 (2010) 2200–2205, http://dx.doi.org/10.1111/j.1365-2672. 2010.04852.x. [29] A. Airoudj, L. Ploux, V. Roucoules, Effect of plasma duty cycle on silver nanoparticles loading of cotton fabrics for durable antibacterial properties, J. Appl. Polym. Sci. (2014) http://dx.doi.org/10.1002/app.41279 (n/a-n/a). [30] F. Hamadi, F. Asserne, S. Elabed, S. Bensouda, M. Mabrouk, H. Latrache, Adhesion of Staphylococcus aureus on stainless steel treated with three types of milk, Food Control 38 (2014) 104–108, http://dx.doi.org/10.1016/j.foodcont.2013.10.006. [31] F.R. Marciano, D.A. Lima-Oliveira, N.S. Da-Silva, E.J. Corat, V.J. Trava-Airoldi, Antibacterial activity of fluorinated diamond-like carbon films produced by PECVD, Surf. Coat. Technol. 204 (2010) 2986–2990, http://dx.doi.org/10.1016/j.surfcoat.2010. 02.040. [32] S. Sobolewski, M.A. Lodes, S.M. Rosiwal, R.F. Singer, Surface energy of growth and seeding side of free standing nanocrystalline diamond foils, Surf. Coat. Technol. 232 (2013) 640–644, http://dx.doi.org/10.1016/j.surfcoat.2013.06.051. [33] F. Pinzari, P. Ascarelli, E. Cappelli, G. Mattei, R. Giorgi, Wettability of HF-CVD diamond films, Diam. Relat. Mater. 10 (2001) 781–785, http://dx.doi.org/10.1016/ S0925-9635(00)00609-9.

[34] V.T. Nguyen, T.W.R. Chia, M.S. Turner, N. Fegan, G.A. Dykes, Quantification of acidbase interactions based on contact angle measurement allows XDLVO predictions to attachment of Campylobacter jejuni but not Salmonella, J. Microbiol. Methods 86 (2011) 89–96, http://dx.doi.org/10.1016/j.mimet.2011.04.005. [35] J. Xia, C. Yang, D. Xu, D. Sun, L. Nan, Z. Sun, Q. Li, T. Gu, K. Yang, Laboratory investigation of the microbiologically influenced corrosion (MIC) resistance of a novel Cubearing 2205 duplex stainless steel in the presence of an aerobic marine Pseudomonas aeruginosa biofilm, Biofouling 31 (2015) 481–492, http://dx.doi.org/10.1080/ 08927014.2015.1062089. [36] C. Kimmel, W. Ballard, S. Kimmel, B. Ullmann, T. Schilling, Stages of embryonic-development of the zebrafish, Dev. Dyn. 203 (1995) 253–310, http://dx.doi.org/10. 1002/aja.1002030302. [37] P.V. Asharani, Y. Lianwu, Z. Gong, S. Valiyaveettil, Comparison of the toxicity of silver, gold and platinum nanoparticles in developing zebrafish embryos, Nanotoxicology 5 (2011) 43–54, http://dx.doi.org/10.3109/17435390.2010.489207. [38] W. Baffone, B. Citterio, E. Vittoria, A. Casaroli, R. Campana, L. Falzano, G. Donelli, Retention of virulence in viable but non-culturable halophilic Vibrio spp, Int. J. Food Microbiol. 89 (2003) 31–39, http://dx.doi.org/10.1016/S0168-1605(03)00102-8. [39] T. Chatterjee, B.K. Chatterjee, D. Majumdar, P. Chakrabarti, Antibacterial effect of silver nanoparticles and the modeling of bacterial growth kinetics using a modified Gompertz model, Biochim. Biophys. Acta Gen. Subj. 1850 (2015) 299–306, http:// dx.doi.org/10.1016/j.bbagen.2014.10.022. [40] S. Wang, C. Yang, L. Ren, M. Shen, K. Yang, Study on antibacterial performance of Cubearing cobalt-based alloy, Mater. Lett. 129 (2014) 88–90, http://dx.doi.org/10. 1016/j.matlet.2014.05.020. [41] J. Luo, H. Liang, L. Yan, J. Ma, Y. Yang, G. Li, Microbial community structures in a closed raw water distribution system biofilm as revealed by 454-pyrosequencing analysis and the effect of microbial biofilm communities on raw water quality, Bioresour. Technol. 148 (2013) 189–195, http://dx.doi.org/10.1016/j.biortech. 2013.08.109. [42] P. Xiangliang, W. Jianlong, Z. Daoyong, Biosorption of Pb(II) by Pleurotus ostreatus immobilized in calcium alginate gel, Process Biochem. 40 (2005) 2799–2803, http://dx.doi.org/10.1016/j.procbio.2004.12.007. [43] H.-C. Flemming, J. Wingender, The biofilm matrix, Nat. Rev. Microbiol. 8 (2010) 623–633, http://dx.doi.org/10.1038/nrmicro2415. [44] J.M.F. Moulder, W.F. Stickle, W.F. Sobol, K.D. Bomben, Handbook of X-Ray Photoelectron Scopy, Perkin-Elmer Corp., Eden Prairie, 1992. [45] O. Sharifahmadian, H.R. Salimijazi, M.H. Fathi, J. Mostaghimi, L. Pershin, Relationship between surface properties and antibacterial behavior of wire arc spray copper coatings, Surf. Coat. Technol. 233 (2013) 74–79, http://dx.doi.org/10.1016/j.surfcoat. 2013.01.060. [46] S. Tang, N. Lu, S.-W. Myung, H.-S. Choi, Enhancement of adhesion strength between two AISI 316L stainless steel plates through atmospheric pressure plasma treatment, Surf. Coat. Technol. 200 (2006) 5220–5228, http://dx.doi.org/10.1016/j.surfcoat. 2005.06.020. [47] G. Gao, D. Lange, K. Hilpert, J. Kindrachuk, Y. Zou, J.T.J. Cheng, M. KazemzadehNarbat, K. Yu, R. Wang, S.K. Straus, D.E. Brooks, B.H. Chew, R.E.W. Hancock, J.N. Kizhakkedathu, The biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides, Biomaterials 32 (2011) 3899–3909, http://dx.doi.org/10.1016/j.biomaterials.2011.02. 013. [48] H. Zhuang, B. Song, V.V.S.S. Srikanth, X. Jiang, H. Schönherr, Controlled wettability of diamond/β-SiC composite thin films for biosensoric applications, J. Phys. Chem. C 114 (2010) 20207–20212, http://dx.doi.org/10.1021/jp109093h. [49] J.H.C. Yang, K. Teii, Mechanism of enhanced wettability of nanocrystalline diamond films by plasma treatment, Thin Solid Films 520 (2012) 6566–6570, http://dx.doi. org/10.1016/j.tsf.2012.06.041. [50] L. Nan, K. Yang, G. Ren, Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus, Mater. Sci. Eng. C 51 (2015) 356–361, http://dx.doi.org/10. 1016/j.msec.2015.03.012. [51] I.T. Hong, C.H. Koo, Antibacterial properties, corrosion resistance and mechanical properties of Cu-modified SUS 304 stainless steel, Mater. Sci. Eng. A 393 (2005) 213–222, http://dx.doi.org/10.1016/j.msea.2004.10.032. [52] Z. He, Q. Wang, Y. Sun, M. Shen, M. Zhu, M. Gu, Y. Wang, Y. Duan, The biocompatibility evaluation of mPEG-PLGA-PLL copolymer and different LA/GA ratio effects for biocompatibility, J. Biomater. Sci. Polym. Ed. 25 (2014) 943–964, http://dx.doi.org/ 10.1080/09205063.2014.914705. [53] L. Tian, G. Zhu, Application of zebra fishes in studies on traditional Chinese medicines, China J. Chin. Mater. Med. 40 (2015) 822–827, http://dx.doi.org/10.4268/ cjcmm20150509.