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A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains
International Biodeterioration & Biodegradation xxx (2017) 1e7
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A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains Wen-Ru Li, Ting-Li Sun, Shao-Lu Zhou, Yong-Kai Ma, Qing-Shan Shi*, Xiao-Bao Xie, Xiao-Mo Huang State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 May 2017 Received in revised form 4 July 2017 Accepted 24 July 2017 Available online xxx
Although both silver ions and silver nanoparticles (AgNPs) have perfect antibacterial activity, but it was assumed that AgNPs have stronger activity than that of silver ions. In this study, we make a comparative analysis of activity, dynamics, and effects of silver ions and two types of AgNPs against four bacterial strains. The minimum inhibitory concentrations (MICs) of silver ions, AgNPs (I) and AgNPs (II) were 0.5, 1 and 2 mg/mL against E. coli, 1, 2 and 8 mg/mL against P. aeruginosa, 1, 2 and 4 mg/mL against S. aureus, and 1, 2 and 2 mg/mL against S. epidermidis respectively. This experimental results showed that Agþ have stronger antibacterial activity than that of AgNPs (I) and AgNPs(II). Antibacterial dynamic curves revealed all the silver ions, AgNPs (I), and AgNPs (II) prolonged the growth lag phase of all four bacteria in a concentration-dependent manner. Furthermore, transmission electronic microscopy (TEM) observation showed that a major part of bacterial cells treated with 2 mg/mL of silver ion and AgNPs were destroyed within 5 h. The transmission electron microscopy (TEM) observation indicated that all the silver ions, AgNPs (I), and AgNPs (II) can induce severe damage in bacterial cells. The flagella of bacteria were damaged or even eliminated, which would cause movement disorders. Many holes or gaps were observed on cell surfaces, which would cause the leakage of cytoplasm and macromolecules, and leading to cell death at last. Our results suggested that silver ions have similar action mode and slightly better antibacterial activity than that of AgNPs against bacterial cells. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Silver ions AgNPs Antibacterial activity Antibacterial dynamics Antibacterial effects
1. Introduction Silver ions and associated compounds are known to act as broad-spectrum antimicrobials and corrosion protection materials. This antimicrobial activity has been recognized since ancient times (Tsezos et al., 1995; Zhao and Stevens, 1998; Feng et al., 2000; Jung et al., 2008; Koizhaiganova et al., 2015), and silver ions have been widely used in the corrosion protection (Silver et al., 2001; Squgo et al., 2015 Mirzaee et al., 2016), medical field for procedures such as dental work, catheterization, and the healing of burn wounds, to control pathogenic bacteria (Jung et al., 2008; Marambio-Jones and Hoek, 2010; Sclocchi et al., 2013; Abdollahi et al., 2015). In addition, a number of chemical forms of silver are known to be good antimicrobials, such as silver nanoparticles (AgNPs) (Kim et al., 2008, 2009; Sanghi and Verma, 2009; Sun et al.,
* Corresponding author. E-mail address: [email protected] (Q.-S. Shi).
2013; Shirakawa et al., 2013), silver sulfadiazine (Klasen, 2000; Silver, 2003), novel composite materials carrying silver (Kawashita et al., 2000; Kim and Kim, 2006; Yoon et al., 2008; Gutarowska et al., 2012a, 2012b; MacMullen et al., 2014). AgNPs, particularly, possess strong antimicrobial properties and have become an important area for research in the antimicrobial field. Many scientists believe that the antibacterial activity of AgNPs is greater than that of silver ions. For example, Lok et al. (2006) reported that the effective antibacterial concentrations of AgNPs and silver ions were in the nanomolar and micromolar ranges, respectively. Surprisingly, our research found that silver ions have good antibacterial activity, sometimes even better than that of AgNPs. Although there have been several studies on the antibacterial effects of silver ions (Feng et al., 2000; Yamanaka et al., 2005; Jung et al., 2008; Rieger et al., 2016) or AgNPs (Sondi and SalopekSondi, 2004; Baker et al., 2005; Sharma et al., 2009; Jiraroj et al., 2014; Franci et al., 2015; Ahmed et al., 2016; Balakumaran et al.,
http://dx.doi.org/10.1016/j.ibiod.2017.07.015 0964-8305/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Li, W.-R., et al., A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains, International Biodeterioration & Biodegradation (2017), http://dx.doi.org/10.1016/ j.ibiod.2017.07.015
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2016; Shirakawa et al., 2016), their mechanism of action underlying these effects, their minimum inhibitory concentration (MIC) and antimicrobial dynamics have not been determined. To elucidate the antibacterial activity, dynamics, and effects of silver ions and AgNPs, and to compare the antimicrobial effects between silver ions and AgNPs, four bacteria strains, E. coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, S. aureus ATCC 6538, and S. epidermidis ATCC 12228, were selected as the research objects of bacteria in the environment for testing. The antibacterial properties of silver ions and two different types of AgNPs were evaluated based on the poisoned food technique, the antibacterial dynamic curves were determined, and cells were observed by transmission electron microscopy (TEM). 2. Materials and methods 2.1. Chemical reagents, microorganisms, mediums, and cultivation Silver nitrate (AgNO3) was purchased from Shanghai Institute of Fine Chemical Materials (Shanghai, China). AgNO3 was suspended in deionized water and the concentration of silver ions was determined by ICP-Mass Spectrometry (Agilent 1260-7700e). Next, a standard solution of Agþ was prepared. The AgNPs solutions of AGS-WMB1000C (I) and AGS-WM 2000 (II) were purchased from Shanghai Huzheng Nanotechnology Company limited (Shanghai, China). The average diameter size of AgNPs (I) and (II) are 5 nm and 20 nm, respectively. The content densities of AgNPs (I) and (II) were 1000 and 2000 mg/mL, respectively. The size and morphology of AgNPs (I) observed by TEM was shown in our previous article (Li et al., 2010), and that of AgNPs (II) is shown in Fig. 1. Bacterial strains E. coli ATCC 8739, P. aeruginosa ATCC 9027, S. aureus ATCC 6538, and S. epidermidis ATCC 12228 were purchased from American Type Culture Collection (ATCC) and were maintained in our laboratory. Mueller-Hinton (MH) medium and Mueller-Hinton agar (MHA) medium, used for aerobic culture of the four bacterial strains at 37 C, were the same as used in our previous work (Li et al., 2014). All solvents and reagents were of analytical grade. 2.2. Antibacterial activities of Agþ, AgNPs (I) and AgNPs (II) against four bacterial strains Antibacterial activity was measured by the poisoned food technique, described in our previous study with slight modifications (Li et al., 2013). The experimental Ag concentrations (mg/mL) of silver ions, AgNPs (I), and AgNPs (II) were all 0 (control), 0.125, 0.25, 0.5, 1, 2, 4, and 8 respectively. The quantity of E. coli,
P. aeruginosa, S. aureus, and S. epidermidis cells in each plate was approximately 106 colony-forming units (CFU). The plates were incubated at 37 C in an incubator for 2 days. The MICs of silver ions, AgNPs (I) and AgNPs (II) against E. coli, P. aeruginosa, S. aureus, and S. epidermidis were determined after incubation for 2 days. Two separate experiments were performed in triplicate. 2.3. Antibacterial dynamics of Agþ, AgNPs (I) and AgNPs (II) against four bacteria strains The antibacterial dynamics of silver ions, AgNPs (I) and AgNPs (II) against E. coli, P. aeruginosa, S. aureus, and S. epidermidis were determined based on methods described in our previous study (Li et al., 2011) with slight modifications. The experimental Ag concentrations (mg/mL) of silver ions, AgNPs (I), and AgNPs (II) used in this study were all 0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, and 8 respectively. Cultures (separately including E. coli, P. aeruginosa, S. aureus, and S. epidermidis) were seeded into 96-well plates at a cell concentration of 106 CFU/mL. Next, each experimental group was incubated at 37 C with shaking at 150 rpm in an automated growth curve analysis system (Bioscreen C). Antibacterial dynamic curves were drawn based on the absorbance at OD600 determined by the automated growth curve analysis system. The experiments were performed in triplicate. 2.4. Morphological alterations of four bacteria treated with Agþ, AgNPs (I) and AgNPs (II) The methods for observing alterations in the morphological structures of the four bacterial strains after exposure to silver ions, AgNPs (I), and AgNPs (II) were the same as described in our previous study (Li et al., 2011, 2013) with slight modifications. The experimental concentrations (mg/mL) of Ag were all 0 (control) and 2. The concentrations of the four bacteria were each 108 CFU/mL. Cultures were incubated at 37 C with 150 rpm in a water bath shaker for 5 h. Then, the bacterial cells were sampled and prepared for TEM (Hitachi H-7650) observation. The experiments were carried out in triplicate. 3. Results and discussion 3.1. Antibacterial activities of Agþ, AgNPs (I) and AgNPs (II) The MICs of Agþ, AgNPs (I), and AgNPs (II) determined by the poisoned food technique, are shown in Table 1. After incubation for 2 d, E. coli colonies filled the control plates, while only dozens of
Fig. 1. Size and morphology observation of AgNPs (II) by TEM (A) and EDX analysis (B).
Please cite this article in press as: Li, W.-R., et al., A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains, International Biodeterioration & Biodegradation (2017), http://dx.doi.org/10.1016/ j.ibiod.2017.07.015
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Table 1 Minimum inhibitory concentration of Agþ and AgNPs against selected microorganisms. Ag Concentration (mg/mL) of Agþ, AgNPs 1 and AgNPs 2 0.125
0.25
0.5
1.0
2.0
4.0
8.0
Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs
1 2 1 2 1 2 1 2 1 2 1 2 1 2
Minimum inhibitory concentration E. coli ATCC 8739
P. aeruginosa ATCC 9027
S. aureus ATCC 6538
S. epidermidis ATCC 12228
þ þ þ þ þ þ e þ þ e e þ e e e e e e e e e
þ þ þ þ þ þ þ þ þ e þ þ e e þ e e þ e e e
þ þ þ þ þ þ þ þ þ e þ þ e e þ e e e e e e
þ þ þ þ þ þ þ þ þ e þ þ e e e e e e e e e
colonies were observed in the experimental group with 0.125 and 0.25 mg/mL of Agþ, and no colonies were observed in the 0.5e8 mg/ mL Agþ groups. After incubation for 2 days, the P. aeruginosa, S. aureus and S. epidermidis colonies filled the control, 0.125, 0.25 and 0.5 mg/mL of Agþ experimental plates, while no colonies were observed in the 1e8 mg/mL Agþ groups. Therefore, the MIC of Agþ was 0.5 mg/mL against E. coli, and 1 mg/mL against the other three strains. It indicated that silver ions had strong antibacterial activity. However, the MIC of AgNPs (I) was 1 mg/mL against E. coli, and 2 mg/ mL against all the other strains. The MICs of AgNPs (II) were 2 mg/ mL against both E. coli and S. epidermidis, 4 mg/mL against S. aureus, and 8 mg/mL against P. aeruginosa. Therefore, the experimental results determined by poisoned food technique indicated that the antibacterial activity of Agþ was always stronger than that of AgNPs (I) and AgNPs (II). The difference in antibacterial activity between the three silver groups was less than 4 times. 3.2. Antibacterial dynamics of Agþ, AgNPs (I) and AgNPs (II) The antibacterial dynamic curves of Agþ, AgNPs (I), and AgNPs (II) against the four bacterial strains are provided in Fig. 2 (E. coli), Fig. 3 (P. aeruginosa), Fig. 4 (S. aureus) and Fig. 5 (S. epidermidis). The growth dynamic trends of the four bacteria in the control were typical growth curves, including a lag phase, an exponential phase, a stabilization phase, and a decline phase. The antibacterial dynamic curves of Agþ (Fig. 2-A), AgNPs (I) (Fig. 2-B), and AgNPs (II) (Fig. 2-C) against E. coli showed that the antibacterial dynamics of AgNPs (I) against E. coli was stronger than that of AgNPs (II) and Agþ. Low concentration of silver in all the three types prolonged the growth lag phase of E. coli. The 0.5 mg/mL of AgNPs (I) could completely inhibited the growth of E. coli. The 1.0 mg/mL of Agþ could play the same inhibitory effect. The 0.5 or 2.0 mg/mL of AgNPs (II) could basically or completely inhibit the growth of E. coli. The antibacterial dynamic curves of Agþ (Fig. 3-A), AgNPs (I) (Fig. 3-B), and AgNPs (II) (Fig. 3-C) against P. aeruginosa showed that the antibacterial dynamics of Agþ against P. aeruginosa was stronger than that of AgNPs (I) and AgNPs (II). The 0.25 mg/mL of Agþ could prolong the growth lag phase of P. aeruginosa, and 0.5 mg/mL could completely inhibited the bacterial growth. However, 2.0 mg/mL AgNPs (I) or 4.0 mg/mL of AgNPs (II) played the same
antibacterial effect. The antibacterial dynamic curves of Agþ (Fig. 4A), AgNPs (I) (Fig. 4-B) and AgNPs (II) (Fig. 4-C) against S. aureus showed the antibacterial dynamics of AgNPs (I) against S. aureus was stronger than that of AgNPs (II) and Agþ. The 1.0 mg/mL of AgNPs (I) could completely inhibited the growth of S. aureus, 2.0 mg/ mL of AgNPs (II) or 4.0 mg/mL of Agþ could play the same antibacterial effect. The antibacterial dynamic curves of Agþ (Fig. 5-A), AgNPs (I) (Fig. 5-B) and AgNPs (II) (Fig. 5-C) against S. epidermidis showed the antibacterial dynamics of Agþ against S. epidermidis was stronger than that of AgNPs (I) and AgNPs (II). The 0.5 mg/mL of Agþ could prolong the lag phase of S. epidermidis to about 50 h, while the same concentration of AgNPs (I) could prolong to about 30 h. Both the 1.0 mg/mL of Agþ and AgNPs (I) could completely inhibited the growth of S. epidermidis. However, the 4.0 mg/mL of AgNPs (II) could play the same antibacterial effect. The experimental results of antibacterial dynamics indicated that the difference in antibacterial activity between the three silver groups was less than 4 times. The antibacterial dynamics of AgNPs (I) against E. coli and S. aureus were stronger than that of AgNPs (II) and Agþ, while the antibacterial dynamics of Agþ against P. aeruginosa and S. epidermidis were stronger than that of AgNPs (I) and AgNPs (II). The antibacterial dynamics of AgNPs (I) is always better than that of AgNPs (II). 3.3. Morphological alterations of bacteria exposed to Agþ, AgNPs (I) and AgNPs (II) Photos of structural alterations of the four bacterial strains exposed to Agþ, AgNPs (I) and AgNPs (II) were observed by TEM and are shown in Fig. 6. Both the control cells of E. coli (Fig. 6-A1) and P. aeruginosa (Fig. 6-B1) showed characteristic features of rodshaped bacteria. Their cellular surfaces were smooth and intact. Moreover, the peripheral flagella of E. coli and the single-end flagella of P. aeruginosa were clear. Similarly, both the control cells of S. aureus (Fig. 6-C1) and S. epidermidis (Fig. 6-D1) showed typical a coccus-shaped morphology. Their cellular surfaces were also smooth and intact. In contrast, after 5 h of exposure to 2 mg/mL silver ions, the E. coli (Fig. 6-A2), P. aeruginosa (Fig. 6-B2), S. aureus (Fig. 6-C2), and S. epidermidis (Fig. 6-D2) cells all showed distinct alterations. The cells had been deformed. Cellular walls were
Please cite this article in press as: Li, W.-R., et al., A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains, International Biodeterioration & Biodegradation (2017), http://dx.doi.org/10.1016/ j.ibiod.2017.07.015
Fig. 3. Antibacterial dynamic curves of Agþ (Fig. 3A), AgNPs (I) (Fig. 3B) and AgNPs (II) (Fig. 3C) against P. aeruginosa.
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Fig. 2. Antibacterial dynamic curves of Agþ (Fig. 2A), AgNPs (I) (Fig. 2B) and AgNPs (II) (Fig. 2C) against E. coli.
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Fig. 5. Antibacterial dynamic curves of Agþ (Fig. 5A), AgNPs (I) (Fig. 5B) and AgNPs (II) (Fig. 5C) against S. epidermidis.
Fig. 4. Antibacterial dynamic curves of Agþ (Fig. 4A), AgNPs (I) (Fig. 4B) and AgNPs (II) (Fig. 4C) against S. aureus.
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Please cite this article in press as: Li, W.-R., et al., A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains, International Biodeterioration & Biodegradation (2017), http://dx.doi.org/10.1016/ j.ibiod.2017.07.015
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Fig. 6. Cellular structural alterations of four bacterial strains exposed to Agþ, AgNPs (I) and AgNPs (II) observed by transmission electron microscopy. (A1) to (D1) show the normal cellular structure of control E. coli (A1), P. aeruginosa (B1), S. aureus (C1), and S. epidermidis (D1) cells; (A2) to (D2) show the structural alterations in E. coli (A2), P. aeruginosa (B2), S. aureus (C2), and S. epidermidis (D2) after exposure to Agþ for 5 h; (A3) to (D3) show the structural alterations in E. coli (A3), P. aeruginosa (B3), S. aureus (C3), and S. epidermidis (D3) after exposure to AgNPs (I) for 5 h; (A4) to (D4) show the structural alterations in E. coli (A4), P. aeruginosa (B4), S. aureus (C4), and S. epidermidis (D4) after exposure to AgNPs (II) for 5 h.
damaged and many holes and gaps were shown on the cellular surface. In addition, the flagella of E. coli and P. aeruginosa had disappeared. After exposure to silver ions for 5 h, the cells of the four bacterial strains were destroyed severely. Similar to silver ions exposure, the cell morphology of the four bacterial species changed irreversibly after exposure to AgNPs (I) (Fig. 6-A3, B3, C3, D3) and AgNPs (II) (Fig. 6-A4, B4, C4, D4). No significantly difference was found in the alteration of cell morphology between the exposure of silver ions, AgNPs (I) and AgNPs (II). This discovery suggested that
the modes of action of silver ions were similar to that of AgNPs in accordance with previous reports (Lok et al., 2006; Rai et al., 2009). Lok et al. (2006) also reported that the mode of action of AgNPs was similar to that of Agþ. The TEM observation indicated that all the silver ions, AgNPs (I) and AgNPs (II) can induce severe damage in bacterial cells. The flagella of bacteria were damaged or even eliminated, which would cause movement disorders. Many holes or gaps were observed on cell surfaces, which would cause the leakage of cytoplasm and
Please cite this article in press as: Li, W.-R., et al., A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains, International Biodeterioration & Biodegradation (2017), http://dx.doi.org/10.1016/ j.ibiod.2017.07.015
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macromolecules, and this could lead to cell death. 4. Conclusions In summary, silver ions have strong antibacterial activity, which are better than that of AgNPs (I) and AgNPs (II). The MIC of Agþ, AgNPs (I) and AgNPs (II) were 0.5, 1, and 2 mg/mL against E. coli, 1, 2, and 8 mg/mL against P. aeruginosa, 1, 2, and 4 mg/mL against S. aureus, 1, 2, and 2 mg/mL against S. epidermidis. Moreover, all the silver ions, AgNPs (I), and AgNPs (II) have good antibacterial dynamics. They can prolong the growth lag phase of bacteria in a concentration-dependent manner. The experimental results indicated the difference in antibacterial activity between the three silver groups was less than 4 times. Furthermore, silver ions and AgNPs can induce irreversible alterations in cellular structure, such as damaged flagella, and holes and gaps on the cellular surface that can cause movement disorders and cell death. The modes of action of silver ions were similar to that of AgNPs. Therefore, silver ions and AgNPs not only have the potential for broad antibacterial applications, but also are very promising antibacterial materials. Acknowledgements Work in the laboratory is funded by grant number 31500113 from the National Natural Science of Foundation of China, 2013B010102014 from Guangdong Province Science and Technology Project, and 201607020020 from Guangzhou Municipal Science and Technology Research Project. References ~ oz, J.A., Tuovinen, O.H., Abdollahi, H., Noaparast, M., Shafaei, S.Z., Manafi, Z., Mun 2015. Silver-catalyzed bioleaching of copper, molybdenum and rhenium from a chalcopyriteemolybdenite concentrate. Int. Biodeter Biodegr 104, 194e200. Ahmed, S., Ahmad, M., Swami, B.L., Ikram, S., 2016. A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: a green expertise. J Adv. Res. 7, 17e28. Baker, C., Pradhan, A., Pakstis, L., Pochan, D.J., Shah, S.I., 2005. Synthesis and antibacterial properties of silver nanoparticles. J. Nanosci. Nanotechnol. 5, 244e249. Balakumaran, M.D., Ramachandran, R., Jagadeeswari, S., Kalaichelvan, P.T., 2016. In vitro biological properties and characterization of nanosilver coated cotton fabrics-An application for antimicrobial textile finishing. Int. Biodeter Biodegr 107, 48e55. Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., Kim, J.O., 2000. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. Res. A 52, 662e668. Franci, G., Falanga, A., Galdiero, S., Palomba, L., Rai, M., Morelli, G., Galdiero, M., 2015. Silver nanoparticles as potential antibacterial agents. Molecules 20 (5), 8856e8874. ra, J., Szynkowska, M., Gliscin ska, E., Gutarowska, B., Rembisz, D., Zduniak, K., Sko g, A., 2012a. Optimization and application of the misting method with Koziro silver nanoparticles for disinfection of the historical objects. Int. Biodeter Biodegr 75, 167e175. Gutarowska, B., Skora, J., Zduniak, K., Rembisz, D., 2012b. Analysis of the sensitivity of microorganisms contaminating museums and archives to silver nanoparticles. Int. Biodeter Biodegr 68, 7e17. Jiraroj, D., Tungasmita, S., Tungasmita, D.N., 2014. Silver ions and silver nanoparticles in zeolite A composites for antibacterial activity. Powder Technol. 264, 418e422. Jung, W.K., Koo, H.C., Kim, K.W., Shin, S., Kim, S.H., Park, Y.H., 2008. Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli. Appl. Environ. Microb. 74 (7), 2171e2178. Kawashita, M., Tsuneyama, S., Miyaji, F., Kokubo, T., Kozuka, H., Yamamoto, K., 2000. Antibacterial silver-containing silica glass prepared by sol-gel method. Biomaterials 21, 393e398. Kim, K.J., Sung, W.S., Moon, S.K., Choi, J.S., Kim, J.G., Lee, D.G., 2008. Antifungal effect of silver nanoparticles on dermatophytes. J. Microbiol. Biotechn 18 (8), 1482e1484. Kim, K.J., Sung, W.S., Suh, B.K., Moon, S.K., Choi, J.S., Kim, J.G., Lee, D.G., 2009.
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A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains Wen-Ru Li, Ting-Li Sun, Shao-Lu Zhou, Yong-Kai Ma, Qing-Shan Shi*, Xiao-Bao Xie, Xiao-Mo Huang State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 May 2017 Received in revised form 4 July 2017 Accepted 24 July 2017 Available online xxx
Although both silver ions and silver nanoparticles (AgNPs) have perfect antibacterial activity, but it was assumed that AgNPs have stronger activity than that of silver ions. In this study, we make a comparative analysis of activity, dynamics, and effects of silver ions and two types of AgNPs against four bacterial strains. The minimum inhibitory concentrations (MICs) of silver ions, AgNPs (I) and AgNPs (II) were 0.5, 1 and 2 mg/mL against E. coli, 1, 2 and 8 mg/mL against P. aeruginosa, 1, 2 and 4 mg/mL against S. aureus, and 1, 2 and 2 mg/mL against S. epidermidis respectively. This experimental results showed that Agþ have stronger antibacterial activity than that of AgNPs (I) and AgNPs(II). Antibacterial dynamic curves revealed all the silver ions, AgNPs (I), and AgNPs (II) prolonged the growth lag phase of all four bacteria in a concentration-dependent manner. Furthermore, transmission electronic microscopy (TEM) observation showed that a major part of bacterial cells treated with 2 mg/mL of silver ion and AgNPs were destroyed within 5 h. The transmission electron microscopy (TEM) observation indicated that all the silver ions, AgNPs (I), and AgNPs (II) can induce severe damage in bacterial cells. The flagella of bacteria were damaged or even eliminated, which would cause movement disorders. Many holes or gaps were observed on cell surfaces, which would cause the leakage of cytoplasm and macromolecules, and leading to cell death at last. Our results suggested that silver ions have similar action mode and slightly better antibacterial activity than that of AgNPs against bacterial cells. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Silver ions AgNPs Antibacterial activity Antibacterial dynamics Antibacterial effects
1. Introduction Silver ions and associated compounds are known to act as broad-spectrum antimicrobials and corrosion protection materials. This antimicrobial activity has been recognized since ancient times (Tsezos et al., 1995; Zhao and Stevens, 1998; Feng et al., 2000; Jung et al., 2008; Koizhaiganova et al., 2015), and silver ions have been widely used in the corrosion protection (Silver et al., 2001; Squgo et al., 2015 Mirzaee et al., 2016), medical field for procedures such as dental work, catheterization, and the healing of burn wounds, to control pathogenic bacteria (Jung et al., 2008; Marambio-Jones and Hoek, 2010; Sclocchi et al., 2013; Abdollahi et al., 2015). In addition, a number of chemical forms of silver are known to be good antimicrobials, such as silver nanoparticles (AgNPs) (Kim et al., 2008, 2009; Sanghi and Verma, 2009; Sun et al.,
* Corresponding author. E-mail address: [email protected] (Q.-S. Shi).
2013; Shirakawa et al., 2013), silver sulfadiazine (Klasen, 2000; Silver, 2003), novel composite materials carrying silver (Kawashita et al., 2000; Kim and Kim, 2006; Yoon et al., 2008; Gutarowska et al., 2012a, 2012b; MacMullen et al., 2014). AgNPs, particularly, possess strong antimicrobial properties and have become an important area for research in the antimicrobial field. Many scientists believe that the antibacterial activity of AgNPs is greater than that of silver ions. For example, Lok et al. (2006) reported that the effective antibacterial concentrations of AgNPs and silver ions were in the nanomolar and micromolar ranges, respectively. Surprisingly, our research found that silver ions have good antibacterial activity, sometimes even better than that of AgNPs. Although there have been several studies on the antibacterial effects of silver ions (Feng et al., 2000; Yamanaka et al., 2005; Jung et al., 2008; Rieger et al., 2016) or AgNPs (Sondi and SalopekSondi, 2004; Baker et al., 2005; Sharma et al., 2009; Jiraroj et al., 2014; Franci et al., 2015; Ahmed et al., 2016; Balakumaran et al.,
http://dx.doi.org/10.1016/j.ibiod.2017.07.015 0964-8305/© 2017 Elsevier Ltd. All rights reserved.
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2016; Shirakawa et al., 2016), their mechanism of action underlying these effects, their minimum inhibitory concentration (MIC) and antimicrobial dynamics have not been determined. To elucidate the antibacterial activity, dynamics, and effects of silver ions and AgNPs, and to compare the antimicrobial effects between silver ions and AgNPs, four bacteria strains, E. coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, S. aureus ATCC 6538, and S. epidermidis ATCC 12228, were selected as the research objects of bacteria in the environment for testing. The antibacterial properties of silver ions and two different types of AgNPs were evaluated based on the poisoned food technique, the antibacterial dynamic curves were determined, and cells were observed by transmission electron microscopy (TEM). 2. Materials and methods 2.1. Chemical reagents, microorganisms, mediums, and cultivation Silver nitrate (AgNO3) was purchased from Shanghai Institute of Fine Chemical Materials (Shanghai, China). AgNO3 was suspended in deionized water and the concentration of silver ions was determined by ICP-Mass Spectrometry (Agilent 1260-7700e). Next, a standard solution of Agþ was prepared. The AgNPs solutions of AGS-WMB1000C (I) and AGS-WM 2000 (II) were purchased from Shanghai Huzheng Nanotechnology Company limited (Shanghai, China). The average diameter size of AgNPs (I) and (II) are 5 nm and 20 nm, respectively. The content densities of AgNPs (I) and (II) were 1000 and 2000 mg/mL, respectively. The size and morphology of AgNPs (I) observed by TEM was shown in our previous article (Li et al., 2010), and that of AgNPs (II) is shown in Fig. 1. Bacterial strains E. coli ATCC 8739, P. aeruginosa ATCC 9027, S. aureus ATCC 6538, and S. epidermidis ATCC 12228 were purchased from American Type Culture Collection (ATCC) and were maintained in our laboratory. Mueller-Hinton (MH) medium and Mueller-Hinton agar (MHA) medium, used for aerobic culture of the four bacterial strains at 37 C, were the same as used in our previous work (Li et al., 2014). All solvents and reagents were of analytical grade. 2.2. Antibacterial activities of Agþ, AgNPs (I) and AgNPs (II) against four bacterial strains Antibacterial activity was measured by the poisoned food technique, described in our previous study with slight modifications (Li et al., 2013). The experimental Ag concentrations (mg/mL) of silver ions, AgNPs (I), and AgNPs (II) were all 0 (control), 0.125, 0.25, 0.5, 1, 2, 4, and 8 respectively. The quantity of E. coli,
P. aeruginosa, S. aureus, and S. epidermidis cells in each plate was approximately 106 colony-forming units (CFU). The plates were incubated at 37 C in an incubator for 2 days. The MICs of silver ions, AgNPs (I) and AgNPs (II) against E. coli, P. aeruginosa, S. aureus, and S. epidermidis were determined after incubation for 2 days. Two separate experiments were performed in triplicate. 2.3. Antibacterial dynamics of Agþ, AgNPs (I) and AgNPs (II) against four bacteria strains The antibacterial dynamics of silver ions, AgNPs (I) and AgNPs (II) against E. coli, P. aeruginosa, S. aureus, and S. epidermidis were determined based on methods described in our previous study (Li et al., 2011) with slight modifications. The experimental Ag concentrations (mg/mL) of silver ions, AgNPs (I), and AgNPs (II) used in this study were all 0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, and 8 respectively. Cultures (separately including E. coli, P. aeruginosa, S. aureus, and S. epidermidis) were seeded into 96-well plates at a cell concentration of 106 CFU/mL. Next, each experimental group was incubated at 37 C with shaking at 150 rpm in an automated growth curve analysis system (Bioscreen C). Antibacterial dynamic curves were drawn based on the absorbance at OD600 determined by the automated growth curve analysis system. The experiments were performed in triplicate. 2.4. Morphological alterations of four bacteria treated with Agþ, AgNPs (I) and AgNPs (II) The methods for observing alterations in the morphological structures of the four bacterial strains after exposure to silver ions, AgNPs (I), and AgNPs (II) were the same as described in our previous study (Li et al., 2011, 2013) with slight modifications. The experimental concentrations (mg/mL) of Ag were all 0 (control) and 2. The concentrations of the four bacteria were each 108 CFU/mL. Cultures were incubated at 37 C with 150 rpm in a water bath shaker for 5 h. Then, the bacterial cells were sampled and prepared for TEM (Hitachi H-7650) observation. The experiments were carried out in triplicate. 3. Results and discussion 3.1. Antibacterial activities of Agþ, AgNPs (I) and AgNPs (II) The MICs of Agþ, AgNPs (I), and AgNPs (II) determined by the poisoned food technique, are shown in Table 1. After incubation for 2 d, E. coli colonies filled the control plates, while only dozens of
Fig. 1. Size and morphology observation of AgNPs (II) by TEM (A) and EDX analysis (B).
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Table 1 Minimum inhibitory concentration of Agþ and AgNPs against selected microorganisms. Ag Concentration (mg/mL) of Agþ, AgNPs 1 and AgNPs 2 0.125
0.25
0.5
1.0
2.0
4.0
8.0
Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs Agþ AgNPs AgNPs
1 2 1 2 1 2 1 2 1 2 1 2 1 2
Minimum inhibitory concentration E. coli ATCC 8739
P. aeruginosa ATCC 9027
S. aureus ATCC 6538
S. epidermidis ATCC 12228
þ þ þ þ þ þ e þ þ e e þ e e e e e e e e e
þ þ þ þ þ þ þ þ þ e þ þ e e þ e e þ e e e
þ þ þ þ þ þ þ þ þ e þ þ e e þ e e e e e e
þ þ þ þ þ þ þ þ þ e þ þ e e e e e e e e e
colonies were observed in the experimental group with 0.125 and 0.25 mg/mL of Agþ, and no colonies were observed in the 0.5e8 mg/ mL Agþ groups. After incubation for 2 days, the P. aeruginosa, S. aureus and S. epidermidis colonies filled the control, 0.125, 0.25 and 0.5 mg/mL of Agþ experimental plates, while no colonies were observed in the 1e8 mg/mL Agþ groups. Therefore, the MIC of Agþ was 0.5 mg/mL against E. coli, and 1 mg/mL against the other three strains. It indicated that silver ions had strong antibacterial activity. However, the MIC of AgNPs (I) was 1 mg/mL against E. coli, and 2 mg/ mL against all the other strains. The MICs of AgNPs (II) were 2 mg/ mL against both E. coli and S. epidermidis, 4 mg/mL against S. aureus, and 8 mg/mL against P. aeruginosa. Therefore, the experimental results determined by poisoned food technique indicated that the antibacterial activity of Agþ was always stronger than that of AgNPs (I) and AgNPs (II). The difference in antibacterial activity between the three silver groups was less than 4 times. 3.2. Antibacterial dynamics of Agþ, AgNPs (I) and AgNPs (II) The antibacterial dynamic curves of Agþ, AgNPs (I), and AgNPs (II) against the four bacterial strains are provided in Fig. 2 (E. coli), Fig. 3 (P. aeruginosa), Fig. 4 (S. aureus) and Fig. 5 (S. epidermidis). The growth dynamic trends of the four bacteria in the control were typical growth curves, including a lag phase, an exponential phase, a stabilization phase, and a decline phase. The antibacterial dynamic curves of Agþ (Fig. 2-A), AgNPs (I) (Fig. 2-B), and AgNPs (II) (Fig. 2-C) against E. coli showed that the antibacterial dynamics of AgNPs (I) against E. coli was stronger than that of AgNPs (II) and Agþ. Low concentration of silver in all the three types prolonged the growth lag phase of E. coli. The 0.5 mg/mL of AgNPs (I) could completely inhibited the growth of E. coli. The 1.0 mg/mL of Agþ could play the same inhibitory effect. The 0.5 or 2.0 mg/mL of AgNPs (II) could basically or completely inhibit the growth of E. coli. The antibacterial dynamic curves of Agþ (Fig. 3-A), AgNPs (I) (Fig. 3-B), and AgNPs (II) (Fig. 3-C) against P. aeruginosa showed that the antibacterial dynamics of Agþ against P. aeruginosa was stronger than that of AgNPs (I) and AgNPs (II). The 0.25 mg/mL of Agþ could prolong the growth lag phase of P. aeruginosa, and 0.5 mg/mL could completely inhibited the bacterial growth. However, 2.0 mg/mL AgNPs (I) or 4.0 mg/mL of AgNPs (II) played the same
antibacterial effect. The antibacterial dynamic curves of Agþ (Fig. 4A), AgNPs (I) (Fig. 4-B) and AgNPs (II) (Fig. 4-C) against S. aureus showed the antibacterial dynamics of AgNPs (I) against S. aureus was stronger than that of AgNPs (II) and Agþ. The 1.0 mg/mL of AgNPs (I) could completely inhibited the growth of S. aureus, 2.0 mg/ mL of AgNPs (II) or 4.0 mg/mL of Agþ could play the same antibacterial effect. The antibacterial dynamic curves of Agþ (Fig. 5-A), AgNPs (I) (Fig. 5-B) and AgNPs (II) (Fig. 5-C) against S. epidermidis showed the antibacterial dynamics of Agþ against S. epidermidis was stronger than that of AgNPs (I) and AgNPs (II). The 0.5 mg/mL of Agþ could prolong the lag phase of S. epidermidis to about 50 h, while the same concentration of AgNPs (I) could prolong to about 30 h. Both the 1.0 mg/mL of Agþ and AgNPs (I) could completely inhibited the growth of S. epidermidis. However, the 4.0 mg/mL of AgNPs (II) could play the same antibacterial effect. The experimental results of antibacterial dynamics indicated that the difference in antibacterial activity between the three silver groups was less than 4 times. The antibacterial dynamics of AgNPs (I) against E. coli and S. aureus were stronger than that of AgNPs (II) and Agþ, while the antibacterial dynamics of Agþ against P. aeruginosa and S. epidermidis were stronger than that of AgNPs (I) and AgNPs (II). The antibacterial dynamics of AgNPs (I) is always better than that of AgNPs (II). 3.3. Morphological alterations of bacteria exposed to Agþ, AgNPs (I) and AgNPs (II) Photos of structural alterations of the four bacterial strains exposed to Agþ, AgNPs (I) and AgNPs (II) were observed by TEM and are shown in Fig. 6. Both the control cells of E. coli (Fig. 6-A1) and P. aeruginosa (Fig. 6-B1) showed characteristic features of rodshaped bacteria. Their cellular surfaces were smooth and intact. Moreover, the peripheral flagella of E. coli and the single-end flagella of P. aeruginosa were clear. Similarly, both the control cells of S. aureus (Fig. 6-C1) and S. epidermidis (Fig. 6-D1) showed typical a coccus-shaped morphology. Their cellular surfaces were also smooth and intact. In contrast, after 5 h of exposure to 2 mg/mL silver ions, the E. coli (Fig. 6-A2), P. aeruginosa (Fig. 6-B2), S. aureus (Fig. 6-C2), and S. epidermidis (Fig. 6-D2) cells all showed distinct alterations. The cells had been deformed. Cellular walls were
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Fig. 3. Antibacterial dynamic curves of Agþ (Fig. 3A), AgNPs (I) (Fig. 3B) and AgNPs (II) (Fig. 3C) against P. aeruginosa.
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Fig. 2. Antibacterial dynamic curves of Agþ (Fig. 2A), AgNPs (I) (Fig. 2B) and AgNPs (II) (Fig. 2C) against E. coli.
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Fig. 5. Antibacterial dynamic curves of Agþ (Fig. 5A), AgNPs (I) (Fig. 5B) and AgNPs (II) (Fig. 5C) against S. epidermidis.
Fig. 4. Antibacterial dynamic curves of Agþ (Fig. 4A), AgNPs (I) (Fig. 4B) and AgNPs (II) (Fig. 4C) against S. aureus.
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Fig. 6. Cellular structural alterations of four bacterial strains exposed to Agþ, AgNPs (I) and AgNPs (II) observed by transmission electron microscopy. (A1) to (D1) show the normal cellular structure of control E. coli (A1), P. aeruginosa (B1), S. aureus (C1), and S. epidermidis (D1) cells; (A2) to (D2) show the structural alterations in E. coli (A2), P. aeruginosa (B2), S. aureus (C2), and S. epidermidis (D2) after exposure to Agþ for 5 h; (A3) to (D3) show the structural alterations in E. coli (A3), P. aeruginosa (B3), S. aureus (C3), and S. epidermidis (D3) after exposure to AgNPs (I) for 5 h; (A4) to (D4) show the structural alterations in E. coli (A4), P. aeruginosa (B4), S. aureus (C4), and S. epidermidis (D4) after exposure to AgNPs (II) for 5 h.
damaged and many holes and gaps were shown on the cellular surface. In addition, the flagella of E. coli and P. aeruginosa had disappeared. After exposure to silver ions for 5 h, the cells of the four bacterial strains were destroyed severely. Similar to silver ions exposure, the cell morphology of the four bacterial species changed irreversibly after exposure to AgNPs (I) (Fig. 6-A3, B3, C3, D3) and AgNPs (II) (Fig. 6-A4, B4, C4, D4). No significantly difference was found in the alteration of cell morphology between the exposure of silver ions, AgNPs (I) and AgNPs (II). This discovery suggested that
the modes of action of silver ions were similar to that of AgNPs in accordance with previous reports (Lok et al., 2006; Rai et al., 2009). Lok et al. (2006) also reported that the mode of action of AgNPs was similar to that of Agþ. The TEM observation indicated that all the silver ions, AgNPs (I) and AgNPs (II) can induce severe damage in bacterial cells. The flagella of bacteria were damaged or even eliminated, which would cause movement disorders. Many holes or gaps were observed on cell surfaces, which would cause the leakage of cytoplasm and
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macromolecules, and this could lead to cell death. 4. Conclusions In summary, silver ions have strong antibacterial activity, which are better than that of AgNPs (I) and AgNPs (II). The MIC of Agþ, AgNPs (I) and AgNPs (II) were 0.5, 1, and 2 mg/mL against E. coli, 1, 2, and 8 mg/mL against P. aeruginosa, 1, 2, and 4 mg/mL against S. aureus, 1, 2, and 2 mg/mL against S. epidermidis. Moreover, all the silver ions, AgNPs (I), and AgNPs (II) have good antibacterial dynamics. They can prolong the growth lag phase of bacteria in a concentration-dependent manner. The experimental results indicated the difference in antibacterial activity between the three silver groups was less than 4 times. Furthermore, silver ions and AgNPs can induce irreversible alterations in cellular structure, such as damaged flagella, and holes and gaps on the cellular surface that can cause movement disorders and cell death. The modes of action of silver ions were similar to that of AgNPs. Therefore, silver ions and AgNPs not only have the potential for broad antibacterial applications, but also are very promising antibacterial materials. Acknowledgements Work in the laboratory is funded by grant number 31500113 from the National Natural Science of Foundation of China, 2013B010102014 from Guangdong Province Science and Technology Project, and 201607020020 from Guangzhou Municipal Science and Technology Research Project. References ~ oz, J.A., Tuovinen, O.H., Abdollahi, H., Noaparast, M., Shafaei, S.Z., Manafi, Z., Mun 2015. Silver-catalyzed bioleaching of copper, molybdenum and rhenium from a chalcopyriteemolybdenite concentrate. Int. Biodeter Biodegr 104, 194e200. Ahmed, S., Ahmad, M., Swami, B.L., Ikram, S., 2016. A review on plants extract mediated synthesis of silver nanoparticles for antimicrobial applications: a green expertise. J Adv. Res. 7, 17e28. Baker, C., Pradhan, A., Pakstis, L., Pochan, D.J., Shah, S.I., 2005. Synthesis and antibacterial properties of silver nanoparticles. J. Nanosci. Nanotechnol. 5, 244e249. Balakumaran, M.D., Ramachandran, R., Jagadeeswari, S., Kalaichelvan, P.T., 2016. In vitro biological properties and characterization of nanosilver coated cotton fabrics-An application for antimicrobial textile finishing. Int. Biodeter Biodegr 107, 48e55. Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., Kim, J.O., 2000. A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J. Biomed. Mater. Res. A 52, 662e668. Franci, G., Falanga, A., Galdiero, S., Palomba, L., Rai, M., Morelli, G., Galdiero, M., 2015. Silver nanoparticles as potential antibacterial agents. Molecules 20 (5), 8856e8874. ra, J., Szynkowska, M., Gliscin ska, E., Gutarowska, B., Rembisz, D., Zduniak, K., Sko g, A., 2012a. Optimization and application of the misting method with Koziro silver nanoparticles for disinfection of the historical objects. Int. Biodeter Biodegr 75, 167e175. Gutarowska, B., Skora, J., Zduniak, K., Rembisz, D., 2012b. Analysis of the sensitivity of microorganisms contaminating museums and archives to silver nanoparticles. Int. Biodeter Biodegr 68, 7e17. Jiraroj, D., Tungasmita, S., Tungasmita, D.N., 2014. Silver ions and silver nanoparticles in zeolite A composites for antibacterial activity. Powder Technol. 264, 418e422. Jung, W.K., Koo, H.C., Kim, K.W., Shin, S., Kim, S.H., Park, Y.H., 2008. Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli. Appl. Environ. Microb. 74 (7), 2171e2178. Kawashita, M., Tsuneyama, S., Miyaji, F., Kokubo, T., Kozuka, H., Yamamoto, K., 2000. Antibacterial silver-containing silica glass prepared by sol-gel method. Biomaterials 21, 393e398. Kim, K.J., Sung, W.S., Moon, S.K., Choi, J.S., Kim, J.G., Lee, D.G., 2008. Antifungal effect of silver nanoparticles on dermatophytes. J. Microbiol. Biotechn 18 (8), 1482e1484. Kim, K.J., Sung, W.S., Suh, B.K., Moon, S.K., Choi, J.S., Kim, J.G., Lee, D.G., 2009.
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Please cite this article in press as: Li, W.-R., et al., A comparative analysis of antibacterial activity, dynamics, and effects of silver ions and silver nanoparticles against four bacterial strains, International Biodeterioration & Biodegradation (2017), http://dx.doi.org/10.1016/ j.ibiod.2017.07.015