Interactions of silver phosphate nanoparticles (SPNPs) and selenium nanoparticles (SeNPs) with Staphylococcus aureus cultures have been studied at the cellular, molecular and protein level. Significant antibacterial effects of both SPNPs and SeNPs on S. aureus were observed. At a concentration of 300 μM, SPNPs caused 37.5% inhibition of bacterial growth and SeNPs totally inhibited bacterial growth. As these effects might have been performed due to the interactions of nanoparticles with DNA and proteins, the interaction of SPNPs or SeNPs with the amplified zntR gene was studied. The presence of nanoparticles decreased the melting temperatures of the nanoparticle complexes with the zntR gene by 23% for SeNPs and by 12% for SPNPs in comparison with the control value. The concentration of bacterial metallothionein was 87% lower in bacteria after application of SPNPs (6.3 μg mg−1 protein) but was increased by 29% after addition of SeNPs (63 μg mg−1 protein) compared with the S. aureus control (49 μg mg−1 protein). Significant antimicrobial effects of the nanoparticles on bacterial growth and DNA integrity provide a promising approach to reducing the risk of bacterial infections that cannot be controlled by the usual antibiotic treatments.
Staphylococcus aureus is often a cause of infection in vascular grafts. Such infection may treated with appropriate antibiotics, but most S. aureus strains have developed resistance to available antibiotics, and resistance even to new antibiotics is steadily growing (van Hal & Fowler, 2013). The use of metal-based drugs and nanoparticles is one of the highly promising means to suppress infections caused by antibiotic-resistant bacteria (Rai et al., 2009). Synthesized silver and selenium nanoparticles (SeNPs) have been found to be bactericidal and have been proved to be good alternatives for the development of antimicrobial agents (Tran & Webster, 2011; Wang & Webster, 2013). Metallic nanoparticles interacting with cellular components (DNA, RNA and ribosomes) deactivate and effectively alter cellular processes (Grigor'eva et al., 2013). It is assumed that silver nanoparticles can penetrate the cell membrane to reach the cytosol due to their ability to dissolve slowly while releasing Ag+ ions, but the exact mechanism of the silver nanoparticles antimicrobial action remains unclear.
Selenium has been studied for various medical applications and as a potential material for orthopaedic implants (Wang & Webster, 2012). There also are studies indicating an ability of selenium compounds to inhibit the growth of bacteria and the formation of bacterial biofilms (Wang & Webster, 2012). Among numerous selenium compounds, 2,4,6-tri-para-methoxyphenylselenopyrylium chloride, 9-para-chlorophenyloctahydroselenoxanthene, and perhydroselenoxanthene have been demonstrated to have antibacterial activity in vitro, especially against S. aureus. However, the precise effect of elemental SeNPs remains largely unknown (Tran & Webster, 2011; Ramos et al., 2012).
In the past decade, study of the toxicological properties of nanomaterials and/or nanoparticles has opened up a new research field known as nanotoxicology (Yan et al., 2013). Research on nanotoxicity is of extremely high scientific, social and economic value (Pumera, 2011). Nanomaterial-induced reactive oxygen species play a key role in cellular and tissue toxicity (Yan et al., 2013). The toxicity of silver nanoparticles is enhanced in the presence of H2O2, highlighting the biological relevance of investigating the oxidative dissolution of these nanoparticles (Ho et al., 2010). In addition, it has been shown that SeNPs can inhibit proliferation and induce apoptosis in promastigote forms of the protozoa Leishmania major (Beheshti et al., 2013).
The aims of this study were to investigate the effects of SeNPs and silver phosphate nanoparticles (SPNPs) on inhibiting growth in S. aureus, morphologically altering the bacteria cell structure and changing its biochemical parameters, and thereby to identify a suitable substance with antibacterial properties to reduce the risk of bacterial (staphylococcal) infections. Another focus was to observe effects of both SeNPs and SPNPs on bacterial metallothionein concentration and to study the interactions of these nanoparticles with the amplified zntR gene.
Materials and methods
Cultivating S. aureus
Staphylococcus aureus (NCTC 8511) was obtained from the Czech Collection of Microorganisms, Masaryk University, CZ. Staphylococcus aureus was inoculated into lysogeny broth medium for 24 h on a shaker at 40 g and 37 °C. The bacterial culture was diluted to OD600 nm = 0.1 for all experiments. Petri dishes were each covered with 3 mL of the S. aureus culture in lysogeny broth that had been grown for 24 h. Discs 1 cm in diameter applied to Petri dishes with bacteria were cut out from vascular grafts produced by the VUP Medical (Czech Republic) and were mixed with SPNPs or SeNPs in various concentrations. Dishes were incubated in a growth chamber at 37 °C for 24 h. Growth was measured by absorbance using a Multiskan EX apparatus (Thermo Fisher Scientific, Germany). For measurement purposes, S. aureus culture was mixed in the microplate with various concentrations of SPNPs and SeNPs. As a control, the culture was also grown without nanoparticles. The concentrations of SPNPs or SeNPs were 0, 10, 25, 50, 75, 150, 225 and 300 μM. Total volume in the microplate wells was always 300 μL (Chudobova et al., 2013a, b).
Analysing biochemical parameters of S. aureus
The effect of nanoparticles on S. aureus was tested using STAPHYtest 24 biochemical detection tests (Erba Lachema, Czech Republic) for the following substances: urease, arginine, ornithine, β-galactosidase, β-glucuronidase, β-glucosidase, phosphatase, esculin, N-acetyl-β-d-glucosamine, sucrose, mannitol, xylose, galactose, trehalose, maltose, mannose, lactose, sorbitol, ribose, fructose, cellobiose, arabinose, xylitol and raffinose. Substances were mixed with S. aureus bacterial culture with or without added nanoparticles (300 μM). Complexes were measured for 24 h using the Multiskan EX apparatus via ascent Software for Multiskan.
Preparing and characterizing SPNPs and SeNPs
SPNPs were prepared by dissolving (di)sodium hydrogen phosphate heptahydrate in water purified to the American Chemical Society (ACS) standard and then adding a solution of SPNPs nitrate in ACS-standard water according to Khan et al. (2012). The reaction proceeded immediately with the formation of yellow colloidal nanoparticles (200–300 nm in diameter; Chudobova et al., 2013a, b). To the prepared SeNPs, sodium selenite pentahydrate Na2SeO3·5H2O (26.3 mg) was dissolved in 50 mL ACS-standard water, and 3-mercaptopropionic acid (40 μL) was then slowly added to the solution under stirring. Afterwards, pH was adjusted to eight using 1 M NaOH. The reaction mixture was stirred for 2 h. SeNPs were stored in darkness at 4 °C. One millilitre of the solution contains 158 μg of Se nanoparticles (50–100 nm in diameter). Chemicals used in this study were purchased from Sigma-Aldrich in ACS purity unless noted otherwise.
SPNPs and SeNPs were measured on a Spectro Xepos spectrometer (Spectro Analytical Instruments, Germany). The samples were measured on a powder diffraction anode X-ray tube at 47.63 kV and 0.5 mA current and were detected with a Barkla scatter aluminium oxide. The sample was measured through the polyethylene bottle side wall 20 mm above the bottom. The spectro xepos software and TurboQuant method were applied for data analysis.
An Olympus IX 71 inverted system microscope (Japan) was used for imaging of the cells. Total magnification was 1600×. The parameters included an exposure time of 32 ms and ISO was set to 200. Structures were also characterized using an FEG-SEM MIRA XMU electron microscope (Tescan, Czech Republic). Samples were coated with 10 nm of carbon using a K950X carbon coater (Quorum Technologies, UK). For automated acquisition of selected areas, the TESCAN image snapper proprietary software tool was used.
Determining total proteins
Pyrogallol red protein assay (Skalab, Czech Republic) is based upon the formation of a blue protein-dye complex in the presence of molybdate under acidic conditions (pH 2.5). 150 μL of the reagent mixture (50 mM succinic acid, 3.47 mM sodium benzoate, 0.06 mM sodium molybdate, 1.05 mM sodium oxalate, and 0.07 mM pyrogallol red) was pipetted into a plastic cuvette. Then, 8 μL of sample was added. Absorbance was measured at λ = 605 nm using a BS-400 chemistry analyser (Mindray, China) after 6 min of incubation. The resulting value was calculated from the absorbance values of the pure reagent mixture and that of the sample after 6 min of incubation.
Differential pulse voltammetric determination of metallothionein
Electrochemical measurements were performed on a 747 VA Stand instrument connected to a 746 VA Trace Analyzer and 695 Autosampler (Switzerland), using a standard cell with three electrodes and cooled sample holder (4 °C). As a supporting electrolyte, Brdicka solution containing 1 mM Co(NH3)6Cl3 and 1 M ammonium buffer [NH3(aq) and NH4Cl, pH 9.6] was used (Adam et al., 2008).
Interaction of zntR gene from DNA with SPNPs and SeNPs
The zntR gene was amplified from S. aureus according to Singh et al. (1999). Spectra were recorded at 200–400 nm after 120 min of interaction using 1-cm quartz cuvettes (Hellma, UK) on a SPECORD 210 spectrophotometer (Analytik Jena, Germany) at 25 °C. Denaturation of a complex of amplified zntR gene with SPNPs and SeNPs in eight concentrations (0, 10, 25, 50, 75, 150, 225 and 300 μM) was monitored spectrophotometrically using a SPECORD S600 spectrophotometer with a diode detector (Analytik Jena). The sample was incubated for 3 min at increasing temperatures at 25–99 °C and the absorbance was measured at 200–800 nm.
Determining protein mass spectra by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)
After cultivation overnight, 500 μL culture (0.1 = OD600 nm) was centrifuged at 14 000 g for 2 min. Supernatant was discarded and the pellet was suspended in 300 μL of deionized water. Then, 900 μL of ethanol was added. After centrifugation at 14 000 g for 2 min, the supernatant was discarded and the obtained pellet was air-dried. The pellet was then dissolved in 25 μL of 70% formic acid (v/v) and 25 μL of acetonitrile and mixed. The samples were centrifuged at 14 000 g for 2 min and 1 μL of the clear supernatant was spotted in duplicate onto an MTP 384 target (Bruker Daltonics) and air-dried at room temperature. Each spot was overlaid with 1 μL of α-cyano-4-hydroxycinnamic acid matrix solution. Spectra were measured on a Bruker MALDI-TOF/TOF apparatus in the m/z range of 2–20 kDa.
Metal nanoparticles caused significant growth inhibition of S. aureus, with application of SeNPs inducing twice as much growth inhibition as SPNPs (7.0 ± 0.5 and 3.0 ± 0.5 mm, respectively). There were also significant morphological changes in the cell structure, these being most evident in the nucleoid (Fig. 1A).
In addition, our synthesized nanoparticles (SPNPs or SeNPs) were characterized with a scanning electron microscope (Fig. 1B). Figure 1(B-d) shows the structure of S. aureus culture without the addition of nanoparticles. In the case of SPNPs, the formations were square or spherical in character (Fig. 1B-e) and in the case of SeNPs only small spherical particles (Fig. 1B-f) were observed, which confirmed our previous hypothesis.
After S. aureus had been exposed for 18 h to SPNPs, inhibition of bacterial growth was observed and absorbance values at 600 nm had decreased from 0.4 to 0.25 a.u. (Fig. 2A-a). In applying the SPNP concentration with the weakest antibacterial activity (10 μM) to the bacterial culture, absorbance values comparable to those of the control group (0.4) were obtained. The greatest decrease in absorbance values (by 38%) in comparison with the control was achieved by applying 300 μM SPNPs (Fig. 2A-a). By contrast, in the case of SeNPs, the minimum inhibitory concentration was achieved when 10 μM SeNPs was applied and the total inhibitory concentration occurred when 300 μM SeNPs was applied (Fig. 2A-b). Half maximal inhibitory concentration values confirmed the obtained results, as IC50 was 268.2 for SPNPs and 4.4 μM for SeNPs.
We also investigated changes in the biochemical parameters of S. aureus after the addition of nanoparticles. Twenty-four hours after application of SPNPs (300 μM), changes in 11 biochemical tests (of 24 tests in total) were observed (Fig. 2B-b). After application of SeNPs, the results of only seven tests were different from those of the control group (Fig. 2B-c).
Because we observed changes in the morphology of the bacterial cell nucleoid after exposure of S. aureus to SPNPs or SeNPs, we investigated the interaction of both nanoparticle types with DNA using amplified zntR gene. After application of the various concentrations of SeNPs (0, 10, 25, 50, 75, 150, 225 and 300 μM) there was a marked increase in absorbance signal at 260 nm with increasing concentrations of SeNPs (Fig. 3A-b) and composition of nanoparticles, but this shift was not observed when SPNPs were applied (Fig. 3A-a). Statistically significant changes were observed in the melting temperatures of the amplified zntR gene in complexes with SPNPs or SeNPs at the highest concentration of 300 μM (Fig. 3B). Differential changes in the melting temperature of amplified zntR gene probably can be caused by different affinity of nanoparticles to the bacterial DNA. A greater decrease in melting temperature in comparison with the control was observed in the interaction of SPNPs and amplified zntR gene, than in the interaction of SeNPs with amplified zntR gene (23% vs. 12%, respectively). SPNPs may have a greater affinity for the amplified zntR gene than SeNPs.
The concentration of bacterial metallothionein was significantly lower (by about 87%) in bacterial cells after application of SPNPs (6.3 μg mg−1 protein) and a higher (about 29%) concentration of metallothionein occurred in bacterial cells after addition of SeNPs (63 μg mg−1 protein) as compared with the S. aureus control (49 μg mg−1 protein; Fig. 3C). The presence of the zntR gene, which encodes metallothionein formation, was confirmed by polymerase chain reaction (results not shown). The effect of nanoparticles probably caused a change in the protein composition of the S. aureus culture, as was demonstrated by MALDI-TOF mass spectrometry, although the change was not statistically significant (Fig. 3D).
Advances in nanotechnology, and particularly in the preparation of metal nanoparticles, can be considered to constitute one of the keys to developing new vascular grafts, because silver ions can lead to the prevention of bacterial infection (Ho et al., 2013). In addition, SeNPs have been found to have high antibacterial activity and they can be easily prepared (Ramos et al., 2012). The antibacterial activity of SeNPs was greater than that of the SPNPs used in this study, and it was comparable to that of the smaller SPNPs (18–21 nm) used by Prabakar et al. (2013). This probably was because we prepared SeNPs that were smaller in size (50–100 nm) than the SPNPs (200–300 nm). The present study showed that the silver nanoparticles exhibited antibacterial activity even at nanoparticle sizes equal to hundreds of nm. When comparing our findings on inhibition of S. aureus growth in the presence of selenium to those from other scientists, we found that our results were similar to those of Naik et al. (2009). However, our colonies were smaller than those of Naik et al., probably because they had used selenium in a complex with 2-selenobenzo[h]quinoline-3-carbaldehyde.
The effect of 40–60 nm SeNPs on S. aureus was studied by Tran & Webster (2011). In that work, bacterial growth was inhibited by SeNPs in the concentration range 7.8–31 μg mL−1 to approximately 1.7% of that seen in the controls and the SeNPs killed approximately 40% of S. aureus cells. Antimicrobial activity of SeNPs was also confirmed in the work of Hariharan et al. (2012).
In our study the highest used concentration of SeNPs (300 μM, 23.7 μg mL−1) caused total growth inhibition within 24 h. Similar results for a 24-h inhibition period in relation to S. aureus growth on selenium nanoparticle-coated polyvinyl chloride are presented in the work of Ramos et al. (2012). The most interesting finding of our study is that the total inhibition concentration of SeNPs should be at least 5 μg mL−1, which corresponds with results published by Tran & Webster (2011). Biochemical parameters showed greater similarity between S. aureus with the addition of SeNPs and the control, but greater differences with the addition of SPNPs.
Our study also demonstrated the interaction of DNA with SeNPs, whereby these particles probably impair the DNA structure of the zntR gene amplified in vitro. This statement is supported by the study of Beheshti et al. (2013), which reported increasing concentrations of SeNPs biosynthesized by Bacillus sp. MSH-1-induced cytotoxic effects and apoptosis in promastigote forms of the protozoa L. major. Apoptosis was demonstrated by DNA fragmentation in the concentration range 1–150 μg mL−1 of SeNPs. A similar toxicity effect of SeNPs on genomic DNA had been reported by Chen et al. (2008) for human melanoma cells treated with chemically synthesized SeNPs. Treatment of A375 human melanoma cells with SeNPs resulted in dose-dependent cell apoptosis, as indicated by DNA fragmentation and phosphatidylserine translocation (Chen et al., 2008).
In our study, the results regarding concentration of metallothionein were comparable to those of Dar et al. (2013). Their group was interested in the production of metallothionein in various microorganisms in response to cadmium and they showed an increased accumulation of cadmium in cells expressing the protein metallothionein in comparison with control cells. Our results show a distinct decrease in metallothionein synthesis after addition of SPNPs to S. aureus. In contrast, only a slight increase was observed after the addition of SeNPs. This may be associated with a similarity in the biochemical parameters of S. aureus with and without the addition of SeNPs. After the addition of SPNPs, S. aureus showed more differences in biochemical parameters. After application of both SPNPs and SeNPs, on the other hand, S. aureus bacterial culture showed only minor differences in MALDI-TOF mass spectra protein fingerprints in comparison with the control. This leads to the conclusion that smaller nanoparticles (SeNPs) have a greater inhibitory effect compared with larger nanoparticles (SPNPs). Our results suggest an effective way to prevent S. aureus infections using nanostructured selenium.
Financial support from Sensor, Information and Communication Systems CZ.1.05/2.1.00/03.0072 is gratefully acknowledged. The authors declare no conflict of interest.