Powerful colloidal silver nanoparticles for the prevention of gastrointestinal bacterial infections

All reagents were of analytical grade and used without further purification. In a typical experiment, 1.7 g (1.0 × 10⁻² mol) of silver nitrate (Aldrich, 99.9%) was dissolved in 100 ml of deionized water. Then the solution of silver nitrate was precipitated with 0.62 g (1.55 × 10⁻² mol) of sodium hydroxide (Aldrich, 99%). The obtained precipitate of silver oxide (Ag₂O) was filtered and dissolved in 100 ml of aqueous ammonia (0.4% w/w, 2.3 × 10⁻² mol) until a transparent solution of silver ammonium complex, [Ag(NH₃)₂]⁺_(aq) was formed. Next, 2.5 g (8.9 × 10⁻³ mol) of oleic acid (Sigma-Aldrich, 99%) was added dropwise into the obtained complex and the resulting solution was gently stirred for 2 h at room temperature until complete homogeneity of the reaction mixture was achieved. Finally, 2 g (1.11 × 10⁻² mol) of glucose was added to the mixture at room temperature with gentle stirring. The reduction process of silver complex solution (in quartz glass) was initiated with UV irradiation. UV treatment was carried out for 8 h under vigorous stirring without additional heating. A UV lamp (λ =  365 nm, 35 W) was used as light source to stimulate the reduction process. Details of the synthesis process can be found elsewhere [25]. The process of silver NPs colloid preparation under UV treatment was controlled by tuning the irradiation time and the pH of the medium. Samples of aqueous dispersion of silver NPs colloid with different silver concentrations in the range from 1 to 10 mg l⁻¹ were prepared for the present study.

The crystalline structure of nanosilver sample was analyzed by x-ray diffraction (XRD, Bruker D5005) using CuKa radiation (λ =  0.154 nm) at a step of 0.02° (2θ) at room temperature. Transmission electron microscopy (TEM, JEOL-JEM 1010) was conducted to determine the morphology and size distribution of the nanosilver. The composition of the nanosilver particles was characterized by energy-dispersive x-ray (EDX-Emax Horiba, S4800 Hitachi). The silver concentration was determined using the method of atomic absorption spectroscopy (AAS, Shimadzu AA-6300).

A feature of absorption spectra of nanosilver particles was measured by UV–Vis absorbance spectra through the formation of an intense and broad band in the visible range which is called the surface plasmon resonance (SPR). Quartz cuvettes with a 10 mm path length were used for the measurement of dispersion spectra.

2.3.1. Bacteria strains and media.

2.3.2. Surface disinfection assay on agar plates.

2.3.3. Turbidity assay in liquid media.

In order to obtain further understanding of the bactericidal and interaction mechanism of the silver NPs, ultrathin sectioning technique was carried out to observe the ultrastructural changes of bacterial cells destroyed by action of the silver NPs. After two strains of bacteria (E. coli, V. cholerae) were exposed to the colloidal silver NPs, the samples were collected and fixed by 2.5% glutaraldehyde in cacodylate buffer (0.1 M) for 30 min at room temperature. The fixed samples were washed by cacodylate buffer three times for 10 min each and transferred to 1% OsO₄/cacodylate buffer for 1 h. The samples were then dehydrated by using a series of alcohol with 50, 70, 80, 90 and 100% (two times × 5 min), and then propylene oxide (three times × 5 min). The samples were infiltrated and finally embedded in Epon 812 at 60 °C for 24 h. The polymerized samples were sectioned into ultrathin slices 60–90 nm in thickness, and placed on collodion-coated copper grids (300 meshes). The analyses of ultrastructural changes of interior of the bacteria cells were conducted by transmission electron microscopy (TEM, JEM 1010, JEOL).

In order to control the process of silver NPs formation we used UV irradiation at the stage of reducing silver ammonium complex. It was found that the UV treatment of the reaction medium containing silver complex, surfactant (oleic acid) and glucose resulted in formation of highly stable dispersion of small (9–10 nm in average size) silver NPs with narrow size distribution [25, 26]. A time period of 8 h was found to be optimal for the complete reduction of silver complex. The UV irradiation causes excitation of [Ag(NH₃)₂]⁺ ions followed by electron transfer from the glucose molecule to Ag⁺, thus producing Ag⁰ atoms which then form clusters and seeds:

$$\begin{eqnarray*} && [{\rm Ag}({\rm NH}_3 )_2 ]^ + + {\rm RCHOH}\buildrel{{h}\nu}\over\longrightarrow {\rm Ag}^0 + 2{\rm NH}_3 + {\rm H}^ + + {\rm R}\dot {\rm COH}, \\ && {{n}}{\rm Ag}^0 \to ({\rm Ag}_{{n}} )^0 , \end{eqnarray*}$$

where RCHOH represents glucose in its cyclic form. The UV treatment leads to the substantially simultaneous formation of a large amount of silver nuclei which then start to grow. This results in small dimensions and narrow size distribution of the finally obtained silver NPs [29, 30].

As evidence, figure 1 shows a UV–Vis spectrum of the colloidal solution of silver NPs at a silver concentration of 10 ppm. As observed from figure 1, an optical absorption band with a maximum at 420 nm was found, which is a typical feature of the absorption of metallic silver NPs due to the surface plasmon resonance (SPR), indicating the presence of silver NPs in the solution. It was found that the as-prepared aqueous dispersions of silver NPs were very stable against aggregation during several months (∼12 months). There is no aggregation behavior of silver NPs as confirmed by the TEM observation (see inset of figure 1). This can be explained by the formation of stabilizing oleate bilayer on the surface of silver particles. Taking into account the stability of the aqueous dispersions of the as-obtained silver NPs, oleic acid most likely forms a double layer between silver surface and liquid medium (figure 2). Since the UV treatment, a large amount of silver nuclei was initially formed. The remaining silver ions get adsorbed on the surface of already formed particles and attract oppositely charged oleate ions. These oleate ions form the first capping layer on the surface of the silver particles (see figure 2). Since the carboxyl group of the oleate ion is directed toward the silver surface, the first capping layer is highly hydrophobic from the outside. Therefore, the oleate ions from the solution start to attach to the first layer forming stable bilayer structure with carboxyl group directed toward liquid medium. Similarly charged bilayer structures protect silver particles from aggregation and, thus, act as particle stabilizers. As a result, this leads to excellent long-term stability of silver NPs colloid prepared, up to several months [25].

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In order to verify highly effective antibacterial activity of as-prepared silver NPs colloid against some infectious gastrointestinal pathogens; two strains of E. coli (ATCC 43888-O157:k-:H7) and V. cholera (O1) were chosen for our present study and these strains were supplied from the National Institute of Hygiene and Epidemiology. Antibacterial activity of the silver NPs colloid against E. coli and V. cholera bacteria was evaluated by means of surface disinfection assay in agar plates, turbidity assay in liquid media [31, 32].

First, we used the surface disinfection method to evaluate the surface antibacterial effect of the silver NPs colloid. For qualitative assessment (by sight) of antibacterial activity agar plates containing silver NPs with different concentrations were inoculated with bacterial suspension 10⁶ CFU. Figures 3(a)–(d) display the observations of bacteria grown on agar plates after 24 h at different concentrations of the silver NPs ranging from 0 mg l⁻¹ (control sample), and from 2 to 6 mg l⁻¹ with counter clockwise direction.

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For E. coli bacteria, it can be seen from figure 3(b) that, for a control sample (0 mg l⁻¹ reaction mixture without adding silver nitrate), the bacteria grown easily within 5 marked circulars on the surface of agar plates. As the silver amount increased, from 2 to 6 mg l⁻¹, the activity of suppression of bacteria growth was increasing as observed from figure 3(b). When the silver content reached 3 mg l⁻¹, a complete inhibition in bacteria growth was observed.

For V. cholera bacteria, it can be seen from figure 3(d) that, at concentrations from 2 to 5 mg l⁻¹ there was evident bacteria growth. When the silver content reached 6 mg l⁻¹, a relatively complete inhibition in bacteria growth was observed. This indicates that the required silver concentration for effective inhibition in bacteria growth against V. cholera bacteria was two times higher than of that against E. coli bacteria. This is due to the difference in biological activity of individual bacterial species. Based on the surface disinfection analysis, it is revealed that the silver NPs colloid displayed noticeable antibacterial effect against E. coli and V. cholera bacteria. This silver NPs colloid prepared is promising for uses as a potential decontaminant agent for controlling bacterial infections.

Next, we further evaluated the antibacterial property of the silver NPs colloid for both E. coli and V. cholera bacteria by measuring the microbial viability of bacteria incubated with the nanosilver. When bacteria (either E. coli or V. cholera) were cultured in LB liquid media containing the nanosilver for 24 h, the mixture containing only LB broth became turbid (see figure 4), this suggests that bacteria in such a mixture medium rapidly proliferated. However, the medium containing the nanosilver remained pellucid (figure 4), indicating few bacteria proliferated. These results showed that the silver NPs colloid prepared could effectively prevent the bacterial growth. It can be seen that at the silver concentration of about 2 mg l⁻¹ the E. coli bacterial growth was inhibited, whereas at the silver concentration over 2 mg l⁻¹ the V. cholera bacterial growth could be prevented. Based on turbidity analysis, we found that the silver concentration of 3 mg l⁻¹ could completely inhibit the bacterial growth for both E. coli and V. cholera bacteria. Our obtained studies indicate that the silver NPs colloid exhibits antibacterial performance in LB liquid media better than that in agar plates media. This result from the increased biological interactions between silver NPs and bacteria in LB liquid media, hence the silver NPs kill tested bacteria more effectively [33].

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In order to provide rudimentary insights into the interaction and bactericidal mechanism between the silver NPs and the E. coli and V. cholera bacteria, ultrastructural and morphological analyses of the electron microscopic technique were conducted. The colloidal solution of silver NPs was dropped onto the surface of E. coli and V. cholera grown on agar plates. After 15, 30 and 60 min, E. coli and V. cholera were taken out and underwent the sectioning method for TEM observation. At different magnifications and sections (figures 5 and 6), many silver NPs bindings around both the E. coli and V. cholera cell membranes as well as inside the cells were found. As observed, the silver NPs first attached to the surface of the cell membrane, penetrating further inside the bacteria. It should be noted that only silver NPs with sufficiently small diameters penetrated into the cells. The cytoplasm was destroyed as the silver NPs penetrated the cell (figures 5(c) and 6(c)). This proves how E. coli and V. cholera cells can be eliminated by the silver NPs.

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The changes took place in the cell membrane morphology, producing a significant increase in their permeability. This affects the proper transport through the plasma membrane, leaving the bacterial cells incapable of properly regulating transport through the plasma membrane, resulting eventually in cell death [34–36]. As shown in figures  5(b) and (d) and  6(b) and (d), in addition to being fixed to the cell membrane, the silver NPs are capable of penetrating through it to be distributed inside a bacterium. After interacting with the E. coli and V. cholera bacteria, the silver NPs adhered to the cell wall of the bacteria and penetrated the cell membrane, resulting in the inhibition of bacterial cell growth and multiplication.

Based on obtained observations (figures 5 and 6), the bactericidal action of our silver NPs was proposed to divide into three stages according to the morphological and structural changes found in the bacterial cells: (i) the silver NPs with sufficiently small diameters attach to the surface of the cell membrane and drastically disturb its proper functions such as permeability and respiration; (ii) then the silver NPs are able to penetrate inside the bacteria and cause further damage by possibly interacting with sulfur- and phosphorus-containing compounds and lose their activity; (iii) inside a bacterium, the silver NPs can interact with DNA, causing the latter to lose its ability to replicate, which may lead to the cell death; finally (iv) the silver NPs release silver ions Ag⁺, which will have an additional contribution to the antibacterial activity of the nanosilver [37].

Here it should be noted that the bactericidal actions of silver NPs (nAg⁰) and silver ion (Ag⁺) are much different from the viewpoint of structural changes. In the case of the silver ions Ag⁺, the antimicrobial action of silver ions is linked with interactions with the thiol group compounds found in the respiratory enzymes of bacterial cells. The silver ions enter into the bacterial cells by penetrating through the cell wall and consequently turn the DNA into condensed form which reacts with thiol group proteins and results in cell death. The silver ions also interface with the replication process. For the case of E. coli, the silver ions act by inhibiting the uptake of phosphate and releasing phosphate, mannitol, succinate, proline and glutamine from E. coli cells [38].

From the application point of view, it is important to check the uses of nanosilver as a potential decontaminant agent for controlling bacterial infections. We conducted a comparative evaluation of disinfection effect of silver NPs colloid and chloramin B (5%) conventionally used in controlling bacterial infections. Figure 7 shows fluorescent images of V. cholera bacteria controlled by our silver NPs and chloramin B (5%) at different treatment time. The obtained results indicate that the silver NPs colloid displayed stronger disinfection effect as compared to the chlolarmin B. At the time of 15 min, the silver NPs colloid showed the highly effective disinfection ability against V. cholera (O1) bacteria. The V. cholera bacteria was mostly destroyed after 60 min when treated by the silver NPs colloid, whereas this bacteria largely remained when treated by chloramin B solution (5%). This reveals that as-prepared silver NPs colloid displayed enhanced antibacterial performance and longer lasting bacterial inactivation efficiency comparative to and better than that of the chloramin B disinfection agent conventionally used.

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With this advantage, the silver NPs colloid is very promising for use in treating environments contaminated with infectious pathogens (bacteria or virus); this will help to effectively prevent and control the outbreak of diseases occurring in the future.