1. Introduction

There are persistent rumors about nanoparticles toxicity and ecotoxicity, concerns about their increasing release into the environment and attempts of their risk assessment [1,2,3,4,5]. Silver nanoparticles (AgNPs) among others (like for example titanium dioxide) belong to the most frequently used ones due to their unique optical, catalytic, sensing and antimicrobial properties. They are declared to be present in consumer products such as cloths and textiles, tooth brushes and pastes, cosmetics, mobile phones and so forth [6,7]. It has been also evidenced in the literature that AgNPs can be spontaneously formed under environmentally relevant conditions [8,9,10]. Thus, there are not only engineered AgNPs which can circulate in the environment.

Antimicrobial properties of AgNPs are often tested employing a standard microdilution method [11] which results in minimum inhibitory concentration (MIC) value determination. Mueller-Hinton broth (M-H Broth) is a standard cultivation medium for many types of bacteria as recommended by Clinical and Laboratory Standards Institute (CLSI), Food and Drug Administration (FDA), European Committee on Antimicrobial Susceptibility Testing (EUCAST), and World Health Organization (WHO) [12]. It comprises amino acids, vitamins, nitrogenous compounds (all three types of compounds stemming from beef infusion), starch and a lot of casein hydrolysate (consisting of proteins rich in oligopeptides). The final pH is 7.4 ± 0.2 at 25 °C [12]. Many of the compounds which are present in M-H Broth can create AgNPs, even though used separately as evidenced in the literature [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29], by reducing Ag⁺ under certain conditions (e.g. alkaline pH, slightly increased temperature etc.). Indeed, there are several papers dealing with AgNPs generation induced by amino acids in alkaline media [17,18,19,20,21], vitamins [22,23], nitrogenous compounds [24,25], proteins [26], polyaminoacids [27], sacharides [28], and starch [29]. Starch, peptides, polyaminoacids and nitrogenous compounds can also serve as good biocompatible capping agents of AgNPs [29,30,31,32,33]. Therefore, there is a real chance that AgNPs are spontaneously formed from Ag⁺ ions in M-H Broth. In the present work, we test this hypothesis which has never been addressed so far. It is of an utmost importance especially when the antibacterial activity of various types of AgNPs is determined and MIC values compared with that of AgNO₃ solution serving usually as a reference.

Silver ions and AgNPs are both well known and used as efficient antibacterial agents [34,35,36,37]. It is generally accepted that Ag⁺ and AgNPs are effective at many bacterial cell levels: they are able to inactivate bacterial enzymes [37,38], disrupt bacterial metabolic processes [39,40,41] and the bacterial cell wall, accumulate in the cytoplasmic membrane and increase its permeability [37,42,43], interact with DNA [38] and generate reactive oxygen species [44,45]. One of the important target sites is the bacterial cell wall which is significantly different in Gram-positive as compared with Gram-negative bacteria. We have thus tested two bacterial strains of both types in the present study, namely: Staphylococcus aureus CCM 3953 (Gram-positive), Staphylococcus epidermidis 879 (Gram-positive), Pseudomonas aeruginosa CCM 3955 (Gram-negative), Klebsiella pneumoniae (ESBL-positive strain) (Gram-negative).

Recent paper of Zong-ming Xiu and coworkers [46] has given clear evidence that Ag⁺ release from AgNPs is the main factor causing AgNPs toxicity. The Ag⁺ release is highly influenced by aerobic conditions, size, shape, and coating of AgNPs [46]. Furthermore, it can be declared that AgNPs coating strongly affects their zeta potential values [33,47]. Surface properties of bacteria species (negative or positive charging) can then come into play as well and result into either favorable, or unfavorable particle-cell electrostatic interactions [35]. Moreover, a study dealing with charge-dependent transport and toxicity of peptide-functionalized AgNPs has been published recently [33]. The authors [33] have demonstrated by the tests using zebrafish embryos that AgNPs with positive zeta potential values are less toxic than those possessing negative values. The peptides employed have differed in a single amino acid at its C-terminus [33]. The most toxic peptide in their work [33] has been ended by L-glutamic acid. For this reason, in the present work we have focused our attention on the influence of L-glutamic acid (E) as a direct surface-modifier of AgNPs. For the sake of comparison, glutamine (Q) in augmented concentrations has been also selected. It is demonstrated that the addition of these two amino acids can change zeta potential values and the other characteristics of engineered as well as spontaneously formed AgNPs.

Characteristic features of AgNPs were determined by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential values measurements, and UV-Vis absorption spectroscopy in the present study. Surface modification of AgNPs by the two selected amino acids was investigated by the methods of vibrational spectroscopy, namely, infrared (IR) absorption and surface-enhanced Raman scattering (SERS).

2. Materials and Method

2.1. Chemicals

Silver nitrate (> 99.0% purity, AgNO₃) purchased from Lachema (Czech Republic) was not purified prior to its use. Sodium borohydride (NaBH₄) purchased from Sigma-Aldrich, stored under humidity-limited conditions (no longer than for half year) was employed. L-glutamic acid (E) and L-glutamine (Q) provided by Sigma-Aldrich were stored under laboratory conditions and used as received. Mueller-Hinton broth (M-H Broth) was purchased from Difco (Becton, Dickinson and Company). All glassware including spectroscopic cuvettes were washed with diluted (1:1 v/v) nitric acid (Penta, Czech Republic) and several times thoroughly rinsed with deionized water.

2.2. Spontaneous formation of AgNPs in M-H Broth

2.35 × 10⁻⁴ M of AgNO₃ was prepared and used as a stock solution for all systems. Then, 3 mL of AgNO₃ stock solution and 3 mL of M-H Broth were mixed together in a testing tube, stored in dark at 37 °C for 24 hours. Characterization by DLS, TEM, UV-Vis was then performed. In the case of systems containing E and/or Q, the system preparations included the addition of E/Q into AgNO₃ solution, the molar ratio between Ag⁺ and AA was hold 1:1 and 1:10.

2.3. Synthesis of borohydride-reduced Ag colloid (Agbh)

Engineered AgNPs were prepared in exactly the same way as described in details in ref. [48]: an aqueous solution (9 mL) of 2.2 × 10⁻³ M AgNO₃ was added drop-wise into 75 mL of 1.1 × 10⁻³ M aqueous solution of NaBH₄ stirred in an Erlenmayer flask placed in an ice-bath. The ice-bath was removed five minutes after the addition of the last drop of silver nitrate solution. Stirring was continued for another 45 min to adjust the Ag colloid to laboratory temperature. Then, 10 min of heating at 40 °C was applied to the Ag colloid while stirring in order to facilitate oxidation of any non-reacted borohydride. Finally, the Ag colloid was allowed to cool down to laboratory temperature with continuous stirring. The resulting Ag colloid was yellow in color, with the maximum of surface plasmon extinction at 390 nm, containing particles of approximately 12 nm in diameter. It was stable for at least 3 months without addition of any stabilizers if stored in dark at room temperature.

2.4. Instrumentation

Transmission electron microscopic images were recorded on a JEOL JEM-2010 TEM. Dynamic light scattering (DLS) and zeta potential values were measured on a Zetasizer Nano Series (Malvern Instruments). UV-Vis spectra were collected on a Specord S600 (Analytic Jena) spectrophotometer by using 1 cm optical path length cuvettes at room temperature. Surface-enhanced Raman scattering (SERS) spectra were recorded on a DXR Raman microscope (Nicolet, Czech Republic) in backscattering mode, using macro-sampler for a 1 cm cuvette, employing the 633 nm excitation wavelength (of a He-Ne gas laser), 8 mW on the sample, 50 μm slit, 4× objective, and high resolution grating (3 cm⁻¹ spectral resolution). Each SERS spectrum was accumulated 100 times for 1 s, baseline corrected in Omnic software and presented without any further smoothing. Infrared (IR) absorption spectra were measured on an iS5 IR spectrophotometer (Nicolet, Czech Republic) using attenuated total reflection (ATR) mode on ZnSe crystal, 64 scans, 2 cm⁻¹ resolution. The IR absorption spectra were treated in Omnic software by the following procedures: subtraction of a background measured immediately before the collection of a sample spectrum, transformation from reflectance to absorbance, baseline correction, and transformation from absorbance to transmittance.

2.5. Antibacterial tests

Antimicrobial activities of the systems were investigated using a standard microdilution method, which has been described in details elsewhere [11]. Minimum inhibitory concentration (MIC) values of AgNPs inducing the inhibition of bacterial growth are determined by this method. The antimicrobial tests were carried out in disposable microtitration plates and repeated three times in duplicates. Four bacterial strains were tested: Staphylococcus aureus CCM 3953 (Czech Collection of Microorganisms, Masaryk University in Brno, Czech Republic), Staphylococcus epidermidis 879 (isolated from the clinical material of a patient of the University Hospital in Olomouc, Czech Republic), Pseudomonas aeruginosa CCM 3955 (Czech Collection of Microorganisms, Masaryk University in Brno, Czech Republic), Klebsiella pneumoniae ESBL-positive strain (isolated from the clinical material of a patient of the University Hospital in Olomouc, Czech Republic).

3. Results and Discussion

3.1. Evidence of spontaneous AgNPs formation from AgNO₃ in M-H Broth

Standard microdilution method for the determination of MIC values [11] was performed for AgNO₃ solution and AgNPs prepared by borohydride-induced chemical reduction (Agbh). Both solutions contained the same initial concentration of Ag⁺, i.e. 2.35 × 10⁻⁴ M. During the antibacterial tests, we observed changes in color for the samples containing AgNO₃ and M-H Broth after 24 hours of incubation with a microbe, while almost no color changes were observed for Agbh under the same experimental conditions—Figure 1.

Figure 1. Evidence for deep color change of the samples containing AgNO₃ and/or AgNO₃ +E (molar ratio being 1:1), M-H Broth and a microbe (Staphylococcus epidermidis 879) when photos taken (A) immediately and (B) after 24 hours of incubation at 37 °C in dark. For the sake of a direct comparison, a sample containing Agbh which was tested under the same conditions is also shown. Wells which TEM images were recorded from are marked.

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Considering the composition of M-H Broth (amino acids, vitamins, nitrogenous compounds, starch, oligopeptides) [12], the conditions of microbes cultivation [11] and taking into account the results published and discussed in the literature [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29], we hypothesized that AgNPs could be formed and aggregated as obvious namely from the wells in the second column, the second and third lines in Figure1B. Therefore, a small amount of the sample (in the second column and the second line in Figure1B) was taken, deposited on a TEM grid, allowed to dry and measured. The microscopic image presented in Figure 2A then confirmed our working hypothesis about spontaneous AgNPs formation in M-H Broth under microbe cultivation conditions. The sample seemed to contain bigger (above 50 nm) and small (below 10 nm) particles, thus a bimodal size distribution (Figure 2A).

Figure 2. TEM images of AgNPs (A) spontaneously formed from AgNO₃ in M-H Broth+microbe medium, while (B) engineered Agbh in M-H Broth+microbe medium. Histograms of intensity-based particle size distribution determined by DLS in solutions of (C) AgNO₃+M-H Broth and (D) Agbh+M-H Broth.

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For the sake of excluding any influence of the TEM sample preparation (drying etc.) on AgNPs formation, we also measured the microscopic images of the sample containing Agbh (engineered NPs) and M-H Broth and a microbe (thus the sample in the first column and the second line in Figure 1B)—TEM image shown in Figure 2B. It should be reminded that Agbh is known to contain almost monodispersed AgNPs of the average size of 10-12 nm in diameter [49,50]. It is thus quite obvious that AgNPs of Agbh remained of the same size as determined before any antibacterial testing and only aggregation was observed.

Dynamic light scattering was also employed in order to check the mean particle size in solutions of (i) AgNO₃+M-H Broth and (ii) Agbh+M-H Broth, after 24 h of their “cultivation” at 37 °C without any microbe in regular laboratory testing tubes (as described in section 2.2). This experiment was performed in order to exclude the possibility of AgNPs formation due to microplates and/or due to electron beam in TEM. The resulting histograms based on light intensity measurements are shown in Figure 2C-D. They revealed bimodal particle size distribution (PSD) for AgNO₃+M-H Broth (Figure 2C) and virtually monodispersed nanoparticles solution in the case of Agbh+M-H Broth (Figure 2D), thus similarly as derived from TEM images (Figure 2A-B). While particles of approximately 340 nm in diameter (88%) and nanoparticles of 30 nm in diameter (12%) could be encountered in the PSD in Figure 2C (i.e., AgNO₃+M-H Broth); nanoparticles of sizes around 17 nm in diameter (99%) dominated the PSD in Figure 2D (i.e., Agbh+M-H Broth). The mean particle sizes determined by TEM and DLS for a particular sample differ due to the well known fact that a contour of metallic particle is observed and visualized by TEM, whereas hydrodynamic diameter including organic envelope and coating around each nanoparticle is determined by DLS measurements. Furthermore, intensity-based PSD of DLS measurements is the only one PSD determined purely experimentally (in comparison to number-based and/or volume-based PSDs), thus without any presumptions about e.g. shape of nanoparticles. On the other hand, intensity-based PSD is strongly influenced by the physical phenomenon that big particles scatter more light than the small ones resulting thus in a seemingly higher percentage of bigger particles. Therefore, the mutual counts of big vs. small particles within a sample have to be considered with a special care.

Another characteristic feature of AgNPs, surface plasmon extinction (SPE) band (extinction = absorption + scattering), was also determined for the above mentioned samples—Figure 3. The solution of AgNO₃+M-H Broth manifested itself by a very broad SPE band with the maximum positioned at around 463 nm and a small shoulder at around 350 nm (black curve in Figure 3A). This shape of SPE band well correlates with the presence of bigger and small particles of different morphologies as determined by DLS measurements and TEM images, respectively. On the contrary, Agbh revealed a narrow SPE band located at ~ 390 nm (black curve in Figure 3B) [48,49,50]. It should be pointed out that the SPE band of AgNO₃+M-H Broth (black curve in Figure 3A) is of a relatively low intensity in comparison to Agbh (black curve in Figure 3B) while the initial concentration of Ag⁺ is the same (2.35 × 10⁴ M) in both cases. This means, in turn, that the efficiency of AgNPs formation in M-H Broth is low (under standard microbe cultivation conditions) and not all AgNO₃ is transformed into AgNPs as confirmed by the presence of a shoulder positioned at 303 nm (black curve in Figure 3A). For the sake of a direct comparison, UV-Vis absorption spectrum of AgNO₃ solution in the employed concentration is also shown in Figure 3B.

Figure 3. UV-Vis spectra of (A) AgNO₃+M-H Broth solutions incubated for 24 h: without (black dotted curve) and with glutamic acid (E, red solid curve, molar ratio of Ag⁺:E is 1:1) and/or glutamine (Q, blue dashed curve, molar ratio of Ag⁺:E is 1:1); (B) Agbh colloid (black dotted curve) and AgNO₃ solution (magenta solid curve).

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Antibacterial testing of the AgNO₃ and Agbh solutions against two selected Gram-positive and Gram-negative bacterial strains (Figure 4) systematically revealed lower MIC values for AgNO₃ than for Agbh (i.e., more toxic AgNO₃ than Agbh). It is in a good agreement with the literature [34] where the authors review and summarize the MIC values of AgNPs vs. AgNO₃ against various types of bacteria (in their Table 3), including bacteria which were tested in the present work. The MIC values of AgNO₃ are always approximately one order of magnitude lower than those of AgNPs of sizes 6-90nm [34]. Why is it so? Although we have evidenced that AgNPs are formed in the system of M-H Broth+AgNO₃, we have also stated above that a remarkable portion of AgNO₃ remained in this system after 24 h. Taking into account the fact that a microbe is introduced, allowed to grow for 24 h and then the test is finished, the concentration of Ag⁺ in the system of AgNO₃+M-H Broth is much higher during the testing period than that being obtained by a partial dissolution of AgNPs in the system of Agbh+M-H Broth. This results in a lower MIC value of AgNO₃ in comparison to Agbh although AgNPs are present in both systems.

Figure 4. MIC values (y axis) of tested solutions (x axis) against two selected (A) Gram-positive and (B) Gram-negative bacterial strains whose names are specified above graphs. Experimental errors of MIC values determination for tested solutions are shown.

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3.2. Influence of augmented concentrations of glutamic acid and/or glutamine on spontaneously formed AgNPs from AgNO₃ in M-H Broth

Since elevated concentrations of amino acids are known to increase the growth of bacteria [51], we started to be interested in the impact of two selected amino acids (E, Q) on AgNPs formed spontaneously in M-H Broth. E and Q were intentionally selected with respect to the results published in [33] and for the sake of a direct comparison, respectively. We assume that these two amino acids may potentially change the growth mechanism of AgNPs in the same M-H Broth used for the tests as in section 1 because they are supposed to interact with AgNPs surfaces by their amino- and carboxylic functional groups. Therefore, such experiments were carried out where the E and/or Q concentrations were adjusted to give the final molar ratio of 1:10 of Ag⁺:AA.

UV-Vis spectra of the systems AgNO₃ +E/Q+M-H Broth, shown already in Figure 3A (for the sake of a direct comparison with the system of AgNO₃+M-H Broth, i.e., without any addition of these two amino acids), revealed narrower SPE bands with the maxima being blue-shifted in comparison to the system of AgNO₃+M-H Broth: AgNO₃+E+M-H Broth possesses the maximum at 438 nm, whereas AgNO₃+Q+M-H Broth manifested itself by the maximum at 408 nm.

Visualization of spontaneously formed AgNPs by TEM images and determination of PSD by DLS measurements of AgNO₃+E/Q+M-H Broth (Figure 5) gave evidence about AgNPs formation with significantly different sizes than those presented in Figure 2A and C (i.e., AgNO₃+M-H Broth). Due to the presence of an increased E concentration, a bimodal size distribution of AgNPs remained (Figure 5C), but the histograms representing two size fractions are much closer to each other than in the case of AgNO₃+M-H Broth (Figure 2C). This is in agreement with morphologies and sizes of AgNPs being mostly encountered in TEM images (Figure 5A). The increased Q concentration also induced big changes in morphologies and sizes of AgNPs: a trimodal PSD was determined by DLS (Figure 5D) and confirmed by TEM imaging (Figure 5B).

Figure 5. TEM images of AgNPs spontaneously formed from AgNO₃ in M-H Broth + microbe medium when (A) glutamic acid and/or (B) glutamine were present (Ag⁺:AA ratio of 1:1). Histograms of intensity-based particle size distribution determined by DLS in solutions of (C) AgNO₃+E+M-H Broth, (D) AgNO₃+Q+M-H Broth.

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Antibacterial tests were performed for these solutions containing AgNO₃+E/Q mixed together with M-H Broth as a cultivation medium. The resulting MIC values (Figure 4) were the same, for both of them, as that of the solution containing solely AgNO₃ and M-H Broth. It means that the increased concentrations of the two selected amino acids did not change MIC values. This gave again indirect evidence that the mode of antibacterial action in the systems containing spontaneously formed AgNPs in M-H Broth lies in the initial Ag⁺ concentration rather than in spontaneously formed AgNPs presence although we can definitely change the growth and consequently characteristic features of these AgNPs by the addition of the increased concentrations of selected amino acids into the cultivation medium. It can be thus summarized that the actual composition of M-H Broth has a tremendous impact on the shape, size, and aggregation state of the spontaneously formed AgNPs.

3.3. Engineered AgNPs (Agbh) surface-modified by glutamic acid and/or glutamine

Taking into account the results of the previous section, the interaction between E and/or Q and engineered AgNPs was tested. Two concentrations of amino acids were employed in order to get the final molar ratios between Ag and amino acid (Ag:AA) of 1:10 and 1:70. First of all, characteristic features of particular colloidal systems were measured, such as UV-Vis spectra, zeta potential values (Figure 6) and AgNPs mean sizes determined by DLS.

Figure 6. UV-Vis spectra of Agbh with the addition of (A) glutamic acid and/or (B) glutamine. Zeta potential values are also mentioned in graphs legend.

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Lower concentration of E in Agbh+E (1:10) system induced an aggregation of AgNPs as can be derived from the appearance of a second maximum located at around 635 nm in the extinction spectrum of this system (Figure 6A, wine dotted curve). On the contrary, the higher E concentration (Agbh+E, 1:70) did not change dramatically the position of SPE band maximum and led only to the occurrence of a shoulder at approx. 460 nm (Figure 6A, olive solid curve). Simultaneously, zeta potential value of the parent Agbh colloid (−38 mV) was changed by the lower and higher E concentrations to 0 mV and +14 mV, respectively. These zeta potential values point to a complete (0mV) and/or partial (+14 mV) destabilization of the systems (i.e., aggregation) which is consistent with the UV-Vis spectra presented in Figure 6A. DLS measurements also corroborated the more pronounced aggregation in Agbh+E (1:10) system by the mean size values of AgNPs being around 1070 nm; whereas approximately 64 nm in Agbh+E (1:70) only.

When glutamine added into Agbh in the mutual molar ratio of 1:10 (Ag:Q), it did not cause a dramatic change in the UV-Vis spectrum (Figure 6B, wine dotted curve) as that observed in the case of glutamic acid (in the same ratio toward Ag). Zeta potential value remained also quite negative (−29 mV) and mean size of AgNPs determined by DLS reached approximately 30 nm; both values reporting about the stability of the system. On the other hand, increasing the Q concentration in the final Agbh+Q system (Ag:Q molar ratio of 1:70) resulted in a broadening of SPE band and appearance of a shoulder at around 580 nm (Figure 6B, olive solid curve). Zeta potential value decreased to −18 mV and DLS mean size augmented to ~ 359 nm.

In order to learn how each of the two amino acids interacts with the surface of AgNPs of Agbh colloid, surface-enhanced Raman scattering (SERS) and infrared (IR) absorption spectroscopies were used (Figure 7). For the former technique, the excitation wavelength of 633 nm was chosen with respect to the broadening of SPE band toward the infrared region and the appearance of the second maximum in the UV-Vis spectra presented in Figure 6. As previously explained, the broadening and occurrence of the second maximum are induced by the aggregation of AgNPs caused by the introduction of amino acids into Agbh colloidal solution. Therefore, information about the interaction of a particular amino acid with AgNPs has to be sought by using the excitation wavelength that matches the secondary maximum and/or shoulder. Then, the same excitation wavelength has to be used for all systems in order to compare them mutually and exclude any changes in spectra caused by molecular resonance. Of course, the absolute SERS spectral intensities (Figure 7A-B) can differ based on the extent of resonance with the excitation wavelength for a particular system. For instance, Agbh+E (1:10) is in the best resonance with 633 nm excitation laser beam, providing thus the best SERS signal (compare Figure 7A and B).

Figure 7. SERS spectra (A, B)—excitation wavelength 633 nm—and IR absorption spectra (C, D) of Agbh+E (A, C) and/or Agbh+Q (B, D) systems of both molar ratios investigated (Ag:AA of 1:10 and 1:70) are shown. Wine (dotted) and olive (dashed) colors (types) of the curves for the lower and the higher AA concentrations, respectively, are held. Only the most intensive and/or important peaks are labeled.

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While SERS spectra revealed very similar relative intensities (within a particular SERS spectrum) and positions of peaks in both cases, Agbh+E as well as Agbh+Q (Figure 7A-B); IR absorption spectra of these systems differed significantly (Figure 7C-D). This discrepancy between the spectra stemming from two vibrational techniques can be easily explained by different selection rules being obeyed for the observation of vibration bands and different physical phenomenon being exploited in each of the two techniques.

The most intensive peak in SERS spectra was located at 241 ± 3 cm⁻¹ (Figure 7A and 6B). It can be attributed to Ag-N covalent bond according to the literature [52]. The peak is, especially at less aggregated systems, broadened by a shoulder positioned at lower Raman shifts values, around 230 cm⁻¹. This can be most probably assigned to Ag-OOC bond [53,54]. The other peaks in SERS spectra (Figure7A-B) can be assigned to borates and polyborates (e.g. 1390 cm⁻¹, stemming from the oxidation of borohydride during Agbh preparation), nitrate anion (e.g. 1045 cm⁻¹, coming from AgNO₃—the starting chemical for Agbh preparation), valence and deformation vibrations of amino acids (e.g. C-C, C-C-C, C-C-H, O-C-O, C-N-H etc. bonds) [52].

IR absorption spectra (Figure 7C and 7D) confirmed the presence of O-H, N-H, aliphatic C-H stretching and deformation bands [52,55]. The characteristic IR absorption peaks of a non-dissociated carboxylic acid positioned at ~ 1730 cm⁻¹ and of a dissociated form (two bands at ~ 1600 and ~ 1400 cm⁻¹) were also detected in many cases (Figure 7C-D) pointing thus to the bonding of amino acids by their carboxylic groups to AgNPs surface [52]. The peak at 1670 cm⁻¹, attributed to NH₂-CO-R vibrational band of glutamine [52,55], increased in its intensity when going from the smaller to the higher ratio of Q:Ag (i.e., from 10:1 to 70:1) which is fully understandable (Figure 7D).

It can be thus summarized that both amino acids are bonded to AgNPs surface through covalent bonds and according to the concentration of a particular amino acid either Ag-N, or Ag-OOC bonds are dominant. This together with borates and their counter ions (Na⁺) strongly influence the final zeta potential value of each system and consequently the AgNPs aggregation state.

MIC values determined for all four Agbh+AA systems (Figure 4) revealed pretty much the same values as those determined for the parent Agbh in the cases of both Gram-positive bacteria included in our study and Pseudomonas aeruginosa. The only difference in MIC value was systematically observed for Klebsiella pneumoniae, a lower MIC value for Agbh than for the other systems with amino acids. These results thus show rather no influence of different zeta potential values of engineered AgNPs (induced by AgNPs surface modification using the two selected amino acids of two different concentrations) on their antimicrobial properties. This can be understood as an indirect proof of the fact that the main toxicity of AgNPs is caused by dissolved Ag⁺ ions rather than AgNPs themselves under the conditions of our antibacterial testing although the exact quantification of silver ratio being present in the form of AgNPs vs Ag⁺ [56] is not experimentally achievable nowadays.

4. Conclusion

The present work demonstrated that AgNPs are spontaneously formed from Ag⁺ ions (introduced as AgNO₃) in M-H Broth under the conditions of microbes cultivation, without and/or with the presence of a microbe. The effect of glutamic acid and/or glutamine on the properties of spontaneously and engineered AgNPs was investigated as well. Due to surface-enhanced Raman scattering and IR absorption spectroscopies, covalent bonding of amino acids to AgNPs surface was evidenced. Characteristic features of AgNPs were changed upon the addition of both selected amino acids, however, the antibacterial activity remained almost unchanged. Antibacterial tests repeatedly revealed a higher toxicity of AgNO₃ (lower MIC values) in comparison to engineered AgNPs, although AgNPs were present in both systems. This can serve as an indirect proof of Ag⁺ ions being the main source of AgNPs antibacterial activity.

Acknowledgments

K. M. S. thanks to the Grant Agency of the Czech Republic for financial support (grant no. P108/11/P657). The authors gratefully acknowledge the support by the project LO1305 of the Ministry of Education, Youth and Sports of the Czech Republic and grant project LF_2013_012. Professors Virender K. Sharma from Texas A&M University, Radek Zboril from RCPTM and Rajender S. Varma from U. S. Environmental Protection Agency are thanked for their comments. The authors acknowledge also to three unknown referees for their time spent reviewing the manuscript and for their valuable comments.

Conflict of Interest

All authors declare no conflicts of interest in this paper.