INTRODUCTION

The widespread infections caused by antibiotic-resistant strains of microorganisms requires us to search for new drugs that have antibacterial activity. Currently, in traumatology and orthopedics, a number of drugs are used that belong to different classes of antibacterial agents. The therapy of patients with infectious complications of periprosthetic infection is often long-term and the transition of the pathological process into a chronic form cannot be ruled out, which necessitates the periodic administration of drugs with antibacterial action [1, 2]. As a result, conditions are created for the formation of strains of microorganisms with multidrug resistance, for which the use of conventional antimicrobials is ineffective.

Recently, a number of studies have shown the possibility of using metal nanoparticles in various fields of medicine [3, 4]. However, the influence of metal nanopowders on the growth and reproduction of microorganisms has remains insufficiently studied. The bulk of the research is devoted to studying the antibacterial activity of metal nanoparticles on reference strains of microorganisms [4], although for practical use it is relevant to study the effect of metal nanoparticles on antibiotic-resistant strains of microorganisms isolated from patients with various pathological processes.

The antimicrobial activity of ultrafine metal powders is determined primarily by their small size, which ensures direct contact with the microscopic surface of the membrane of a living object [5]. A number of studies have shown that metal nanoparticles interact with a bacterial cell in several stages. At the first stage, nanoparticles are attached to the surface of the microorganism due to the arising electrostatic voltage [6, 7]. Further, nanoparticles penetrate inside, which leads to changes in the cell membrane, an outflow of the intracellular matrix and, as a result, death of the organism’s cell [5, 7–9].

The study of the antibacterial action of metal nanoparticles is devoted mainly to ultrafine powders of silver and copper. There are few publications on the effect of nickel nanoparticles on the standard strains. Escherichia coli, Lactobacillus ssp., Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis [10–12]. Moreover, in some cases, nickel nanoparticles showed more pronounced antibacterial properties than silver nanoparticles [13]. Studies on the toxicity of nickel nanoparticles in experimental animals have shown that it is lower than that of silver nanoparticles [14].

The antimicrobial properties of metal nanoparticles directly depend on their physicochemical characteristics [15]. Smaller nanoparticles have a greater adsorption capacity and, as a result, more pronounced activity against biological objects. The prospect of using metal nanoparticles as antibacterial drugs has created the need for careful standardization, since any change in the characteristics leads to a change in the antimicrobial activity.

MATERIALS AND METHODS

We used nickel nanoparticles (TU 1733-056-00209013-2008), synthesized at the plasma chemical complex of the State Research Institute of Chemistry and Technology of Organoelement Compounds, a branch of the Federal State Unitary Enterprise of the Russian Federation State Research Center GNIIKhTEOS, Moscow. The nanoparticles were obtained from large metal samples using plasma technology based on the evaporation of raw materials into ultrafine particles of the required size in a plasma stream at a temperature of 5000 to 6000 K and vapor condensation.

The atomic composition of the surface of nickel nanoparticles was determined with a SEM PHI-4300 scanning Auger microprobe (PC-Service, Germany). This study is based on the determination of the structure of matter by measuring the energy spectrum of Auger electrons emitted due to the internal energy conversion of a beam of high-energy electrons with which the sample was irradiated. The qualitative electronic composition of the test sample was determined from the energy spectrum of Auger electrons and the quantitative content of atoms in the sample was determined by the value of their current.

The ζ potential and average hydrodynamic size of nickel particles were measured by dynamic light scattering. For the study, a suspension of nickel nanoparticles in Milli-Q deionized water (Millipore, France) was prepared. The final concentration of nickel nanoparticles was 0.5 mg/mL. Immediately after the preparation of the samples, measurements were performed on a Malvern Zetasizer Nano (Malvern, UK) according to the standard methods.

The ζ potential of bacterial cells was measured before  and after exposure to a nanosized metal powder. Then, 100 μL of a suspension containing 300 000 microbial bodies/mL was added to the suspension of nickel nanoparticles. Measurements were performed 30 minutes after preparing the suspension.

The exact size and morphological characteristics of the nickel nanoparticles were studied using a Tescan Mira II LMU (Tescan, Czech Republic) scanning electron microscope (SEM) with a resolution of 3 nm.

Forty clinical strains with multiple antimicrobial resistance were used as test strains to study the antibacterial activity of the nickel nanoparticles; 20 of them were related to E. coli; and the remaining 20, to S. epidermidis. Microbial cultures were isolated from patients with infectious complications after the arthroplasty of large joints treated at the National Research Institute of Traumatology, Orthopedics, and Neurosurgery, Federal State Budgetary Educational Institution of Higher Education (FSBEI HE), Razumovsky Saratov State Medical University, Far Eastern Branch, Russian Academy of Sciences, Russian Ministry of Health.

The antibacterial activity of nickel nanoparticles at concentrations of 0.01, 0.05, 0.1, 0.5, and 1 mg/mL were studied. A suspension of metal nanoparticles was prepared in a 0.9% NaCl solution. One-hundred μL of a suspension of the studied microorganisms was added to a test tube containing 900 μL of a suspension of nickel nanoparticles. The final concentration of microbial bodies in each tube was 30 000 CFU/mL. Samples were incubated for 30, 60, 90, and 120 min at a temperature of 37°C. The samples were sewn on a dense agar medium Agar nutrient (Becton Dickinson, United States) and thermostated for 24 hours at 37°C. Next, the colonies that had grown were counted. A suspension of microorganisms in a 0.9% NaCl solution served as the control.

The percentage of reduced microorganisms was calculated (% reduction) for the further analysis of the data obtained [16]:

$${\text{Reduction}}{\text{,} \% } = 100 - \left( {\frac{{NT \times 100}}{{NK}}} \right),$$

where Nk is the number of microbial cells in the control and NT is the number of microbial cells in the experiment.

The biochemical properties of the clinical strains of the microorganisms after exposure to nickel particles were changed using the set of ENTEROtest16 and STAFItest16 (La Chema, Czech Republic) reagents in accordance with the instructions attached to the set.

The statistical data processing was carried out using Microsoft Excel 2010 and Statistica 10. When planning the experiment, a power analysis was performed. To assess the normality of the distribution of the quantitative indicators, the Kolmogorov–Smirnov criterion was used. It was established that all the variables included in the study corresponded to the normal distribution. As a result, they considered it possible to use them for assessing differences between samples of Student’s t-criterion. Differences between the qualitative traits were evaluated using the Pearson consent criterion (χ2) corrected by Yates for continuity. The differences were considered significant when the probability value p was <0.05.

RESEARCH RESULTS AND DISCUSSION

The chemical composition, size, shape, and ability to form agglomerates are important characteristics that determine the antimicrobial activity of metal nanoparticles [14]. Hence, the study of the basic physicochemical characteristics of nickel nanoparticles is relevant. The data obtained make it possible to accurately characterize metal nanoparticles, which is necessary for their further study and application.

The chemical composition of nickel nanoparticles was analyzed by Auger electron spectroscopy. The study identified the atomic fraction of elements in the composition of the surface of metal nanoparticles (Fig. 1). The data obtained provide important information on the presence of additional elements that make up the nanopowder. As a result of the study, 64.15% Ni, 7.72% O, 12.29% S, and 15.84% Be were found in the composition of nickel nanoparticles. The presence of oxygen in the composition of the surface of the surface is an indirect confirmation of the presence of an oxide shell, which ensures a gradual release of the ions of the substance and thereby prolongs their active state.

Fig. 1.
figure 1

(Color online) Atomic fraction of nickel elements in the composition of the surface of nickel nanoparticles.

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Research on the ζ potential allows us to determine the physical features of the presence of metal nanoparticles in a solution, and in particular, their ability to form associates of various sizes. As is well known, the magnitude of the ζ potential is proportional to the charge of the colloidal particle. Consequently, with its increase, the ability of metal nanoparticles to form associates declines. In this case, the ζ-potential was +3.51 mV. The obtained values were lower by a factor of 10 than the threshold indicator (±30 mV), which ensures the stability of the colloidal system. The average hydrodynamic size of nickel nanoparticles is determined. The size of this indicator was 1145.00 ± 89.60 nm. Therefore, the nickel nanoparticles included in the study have the ability to form large associates due to the strong chemical bonds that hold the nanoparticles together. This is confirmed by the electron microscopy studies. The resulting images are presented in Fig. 2.

Fig. 2.
figure 2

SEM image of nickel nanoparticles.

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Analysis of the SEM images revealed the presence of granular structures (nanoparticles) tightly connected to each other. The size of individual particles was 80.51 ± 2.21 nm.

One of the important biophysical characteristics of the cells of living organisms is the charge on their surface (ζ-potential). As is well known, a bacterial cell carries a negative charge on its surface, which is associated with the structural features of the cell wall and the presence of lipid-containing compounds in the molecules. As a result of the study, a positive value of the ζ potential for nickel nanoparticles (+3.51 mV) was established. The data obtained confirm the possibility of the occurrence of electrostatic voltage between the surface of a bacterial cell and metal nanoparticles and, as a consequence, the development of an antibacterial effect.

In this work, we determined the ζ-potential of bacterial cells before exposure to metal nanoparticles. As a result, the value of this indicator for the S. epidermidis  strains  was –19.1 ± 1.67 mV; and for E. coli, –41.05 ± 2.61 mV. As can be seen, for the cells of Gram-negative microorganisms, the ζ-potential value was lower, which is related to the presence in the cell wall of a large number of lipid-containing compounds that make up the lipopolysaccharide layer (LPS). During the joint incubation of bacterial cells with metal nanoparticles for 30 min, a significant shift of the studied parameter towards a positive value was observed. After exposure to nanoparticles, for the S. epidermidis strains the ζ-potential was –7.3 ± 0.54 mV; and for E. coli, –32.13 ± 2.13 mV.

Thus, the primary interaction of metal nanoparticles with prokaryotic cells occurs due to the electrostatic interaction of positively charged metal nanoparticles with the negatively charged surface of the bacterial cell. As a result of the study, it a dependence of the changes in the ζ potential on the structure of the cell wall of the microorganisms after exposure to nickel nanoparticles was not established.

The effect of nickel nanoparticles on the clinical E. coli strains was studied (Fig. 3). The low doses of nickel nanoparticles (0.01 and 0.05 mg/mL) caused a decrease in the number of microorganisms capable of growth on solid nutrient media. An increase in the number of nickel nanoparticles led to an increase in the antibacterial effect. Thus, the maximum concentration of nanosized metal powder (1 mg/mL) contributed to the almost complete lysis of the bacterial cells. At an exposure time of 120 min, only 8.62 ± 1.64% of the microorganisms did not lose the ability to grow on a solid nutrient medium.

Fig. 3.
figure 3

Antimicrobial effect of nickel nanoparticles on E. coli strains.

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The effect of nickel nanoparticles on the clinical S. epidermidis strains was studied (Fig. 4). After exposure to metal nanoparticles at a concentration of 0.01 mg/mL, the number of microorganisms that grew on a solid nutrient medium was statistically significantly (p < 0.05) lower than in the control group. The action of nickel nanoparticles over a period of 120 min contributed to the reduction of viable microbial bodies to 15.38 ± 1.35% (p < 0.001). The increase in the dose of the nickel nanopowder led to an increase in the antibacterial effect: starting from a concentration of 0.5 mg/mL and an exposure time of 120 min, there was a complete absence of growth of microorganisms on the agarized nutrient medium.

Fig. 4.
figure 4

Antimicrobial effect of nickel nanoparticles on S. epidermidis strains.

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Analyzing the data obtained, we can conclude that antibacterial activity of nickel nanoparticles was higher than that of the clinical antibiotic-resistant S. epidermidis and E. coli strains. As can be seen from the diagrams (Figs. 3, 4), there is a clear correlation between the activity of metal nanoparticles, the concentration of nanoparticles in a suspension, and the exposure time.

Based on the structural features of the cell wall, a number of studies have shown the dependence of the antibacterial activity of metal nanoparticles on the taxonomic affiliation of bacterial cells [17, 18]. According to the results of the study, it can be concluded that nickel nanoparticles had a stronger effect on the cells of Gram-positive microorganisms (clinical S. epidermidis strains). As can be seen from Table 1, exposure to 0.5 mg/mL of nickel nanoparticles for 120 min caused the complete lysis of the pathogen cells. The percentage of reduced cells after exposure to nickel nanoparticles on the Gram-negative E. coli strains did not reach 100%; the maximum value was 91.38 ± 1.64% (at a concentration of 1 mg/mL, exposure time of 120 min). As we know, the composition of the outer membrane of Gram-negative bacteria cells includes LPS, which is an additional barrier that does not allow substances to penetrate inside.

Table 1. Reduction of strains of Gram-positive and Gram-negative strains of microorganisms after exposure to nickel nanoparticles
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All metabolic processes within the cell take place with the participation of enzymes. Thus, we need to study the effect of nickel nanoparticles on the metabolism of microorganisms. As a result of the study undertaken by us, the effect of nickel nanoparticles on the biochemical processes taking place inside E. coli cells statistically significantly (χ2 = 9.64, p = 0.0019) established a decrease in the number of pathogen strains with the ability to hydrolyze esculin. After exposure to metal nanoparticles on S. epidermidis cells, we observed a decrease in the number of strains capable of reducing nitrates to nitrites (χ2 = 6.29, p = 0.0125). The obtained data can be regarded as the response of a microorganism cell to external exposure.

CONCLUSIONS

As a result of the study, the main physicochemical characteristics of nickel nanoparticles obtained at the plasma chemical complex of the State Research Institute of Chemistry and Technology of Organoelement Compounds, a branch of the Russian Federal State Unitary Enterprise, Moscow, were used. The ζ potential for nickel nanoparticles was 3.51 mV. It was found that metal nanoparticles are capable of forming large agglomerates (1145.00 ± 89.60 nm), which is an indirect confirmation of the presence of the strong chemical bonds that hold the particles together. The ability of metal nanoparticles to aggregate is an important physical characteristic that affects the severity of antibacterial activity. Scattered metal nanoparticles have a different reactivity and surface reaction rate with respect to biological objects, which reduces their antimicrobial activity [18]. In addition, the size of individual nanosized particles of nickel was determined to be 80.51 ± 2.21 nm.

On the surface of metal nanoparticles, the presence of an oxide film was found to provide a prolonged action of the substance. In addition, the presence of oxygen in the composition of the surface of metal nanoparticles is one of the aspects of their toxicity and, as a consequence, the severity of their antimicrobial activity. In the composition of the surface of nickel nanoparticles, 64.15% nickel, 7.72% oxygen, 12.29% sulfur, and 15.84% beryllium were found.

The indicators of the ζ-potential of the cells of microorganisms before and after exposure to metal nanoparticles were studied. The data obtained confirm the adhesion of metal nanoparticles on the surface of the microbial cell due to the occurrence of electrostatic voltage. This stage of interaction, apparently, does not lead to the death of the microorganism but only ensures the close interaction of metal nanoparticles with the bacterial cell.

The antibacterial action of nickel nanoparticles with the physicochemical characteristics described above in clinical antibiotic-resistant strains of Gram-positive (S. epidermidis) and Gram-negative microorganisms (E. coli) isolated in patients with implant-associated infections was studied. The dependence of the antimicrobial activity of nickel nanoparticles on the concentration and duration of exposure was revealed. The highest activity of nickel nanoparticles was observed in relation to clinical S. epidermidis strains, which may be due to the structural features of the cell wall. The lipopolysaccharide layer of Gram-negative microorganisms creates a barrier to the penetration of substances of exogenous origin into the cell.

The effect of nickel nanoparticles on the cell metabolism of Gram-negative and Gram-positive microorganisms was assessed. A decrease in the saccharolytic activity of clinical E. coli strains after exposure to nickel nanoparticles, as well as a decrease in the number of S. epidermidis strains capable of reducing nitrates to nitrites, was established.

Changing the environmental conditions for a bacterial cell is a stress factor. The initial response to stress is aimed at compensating the shift in the internal equilibrium, which ensures the survival of the microorganism. In almost all cases, this answer is based on the existing biochemical mechanisms. Changes in the expression of the genes responsible for the synthesis of new components of the bacterial cell may also occur [19].

The results obtained provide a prerequisite for further studying the use of nickel nanoparticles as antibacterial agents for the treatment of infectious and inflammatory complications in traumatology and orthopedics caused by microorganisms, mainly existing in biofilm form, resistant to most antimicrobial agents.