Antibacterial activity of biosynthesized silver nanoparticles against Pseudomonas aeruginosa

Introduction 

Infectious diseases were the second leading cause of death worldwide in 2023 [1]. Antibiotic resistance became a major problem associated with microbial infections in recent years [2, 3]. Pseudomonas aeruginosa is among of the most dangerous opportunistic microorganisms with multiple drug resistance. Globally, over the past two decades, Pseudomonas aeruginosa was one of the leading agents in the development of nosocomial infections, especially in patients with postoperative surgical trauma, in intensive care units, burn and trauma units, as well as in patients with pre-existing conditions such as cystic fibrosis [4]. P. aeruginosa was classified as one of the most life-threatening infections by the World Health Organization and was included in the list of priority pathogens for research and development of new antibiotics [5]. Resistance of Pseudomonas aeruginosa to antibiotic-like substances is mediated by a complex of composite mechanisms, such as increased expression of the efflux pump, genetic determinants, targeted genome modification systems, and enzymatic systems leading to inhibition of action and inactivation of antimicrobial drugs [6]. Multiple drug resistance is currently the main problem encountered in the development of antibacterial agents. This is also true because the effectiveness of a substance is mainly associated with one of the mechanisms of its action on the bacterial cell. High resistance of P. aeruginosa to various antibiotics necessitates the development of alternative antimicrobial drugs that can effectively overcome the protective barriers of the bacterial cell due to a combination of different mechanisms of action. Nanomaterials are possible candidates for this role [1, 7]. Currently, metal nanoparticles, in particular silver nanoparticles (AgNPs), are considered the most promising agents capable of overcoming multiple antibiotic resistance due to their multidirectional mechanisms of action [8].

We previously discovered the ability of soil nonpathogenic associative bacterial species, Azospirillum brasilense, to biosynthesize gold and silver nanoparticles when cultivated in a liquid nutrient medium in the presence of chloroauric acid and silver nitrate, respectively [9]. Biologically synthesized nanoparticles are more promising for biomedical use than chemically synthesized particles due to their biocompatibility and functionalized surface [10].

Objective – to analyze the antibacterial effect of biosynthesized silver nanoparticles on reference and clinical strains of P. aeruginosa in vitro.

Materials and Methods 

The A. brasilense SR80 strain obtained from the collection of nonpathogenic microorganisms owned by the Institute of Biochemistry and Physiology of Plants and Microorganisms (IBPPM) of the Russian Academy of Sciences (www.collection.ibppm.ru) was used for the green synthesis of silver nanoparticles. This strain is characterized by increased phenoloxidase activity [11]. The bacteria were cultured in a liquid synthetic malate medium with pH=6.8. A 36-h bacterial culture was used in the experiment. The cells were precipitated by centrifugation for 15 min at g=7,000. The supernatant was used in reactions of nanoparticle biosynthesis. The latter was performed by reducing silver nitrate with a 24-h incubation of an aqueous solution of 1 mM of AgNO3 (Dia-m, Russia) and the obtained cell-free extract in the dark at room temperature. A Specord 250 spectrophotometer (Analytik Jena, Germany) was employed to measure absorption spectra. The size, shape, and relative amount of electron-dense nanoparticlesv were assessed using transmission electron microscopy (TEM) images obtained on a Libra 120 microscope (Carl Zeiss, Germany). A Zetasizer Nano ZS device (Malvern, UK) was used to measure the zeta potential, average size, and size distribution of the synthesized particles. We determined the antimicrobial activity of AgNPs against the reference strain of P. aeruginosa ATCC 27853 obtained from the collection of living cultures at the Department of Microbiology, Virology and Immunology, Saratov State Medical University of the Russian Federation Ministry of Healthcare, and against the clinical strain of P. aeruginosa isolated in the bacteriological laboratory of Saratov Regional Children’ Clinical Hospital from the upper respiratory tract of children undergoing treatment. The clinical strain was identified by the test systems for biochemical identification, STAPHYtestT24 and NEFERMtest24 (Erba Lachema, Czech Republic). All strains were grown on pepted meat broth (State Research Center for Applied Microbiology and Biotechnology, Russia). 

Quantitative assessment of antibacterial activity (expressed as the level of relative respiratory activity) was determined by the fluorimetric resazurin test: 100 μL of a 24-hr bacterial culture (cell concentration 1×105 CFU/mL), 50 μL of alamarBlue solution (Sigma, USA) with a concentration of 1 mg/mL and 50 μL of nanoparticles resuspended in phosphate buffer were added to the wells of a 96-well cell culture plate. The plates were incubated at 37 ºC for 24 hours, then the fluorescence intensity was recorded on the Agilent Cary Eclipse fluorescence spectrometer (USA). We would like to point out that respiratory activity of the 24-h bacterial culture was assumed to be 100%. 

The results of quantitative tests were processed using the Microsoft Office Excel software. To assess the statistical significance of differences between samples in the experimental and control experiments, the Student’s t-test was employed. Based on the results of calculating the arithmetic mean and standard deviation for a given sample size n (the number of measurements), the standard error of the mean and the boundaries of its confidence interval were determined taking into account the Student’s t-coefficient at a significance level of 95% (p=0.05) and a sample size, n=6.

Results

When the supernatant obtained from culturing the A. brasilense SR80 strain was incubated with an aqueous solution of AgNO3 in the concentration range from 0.05 to 50 mM, the culture medium acquired a yellow color, which was becoming yellow-brown over time. This color implied the accumulation of silver nanoparticles in the culture medium (Figure 1).

The formation of silver nanoparticles of irregular spherical shape with a size of 5–30 nm was confirmed by transmission electron microscopy. Further incubation led to an increase in the yield of the formed particles without changing their geometric parameters. An increase in the concentration of silver nitrate led to a change in the shape of the nanoparticles from regular spherical to irregular spherical, with sizes ranging 6–35 nm (Figure 2). Data on the size distribution of biosynthesized nanoparticles obtained by transmission electron microscopy were confirmed by the dynamic light scattering method. The zeta potential of silver nanoparticles obtained with different biosynthesis parameters was –17.5±4 mV, which indicates the electrostatic stability of the particles and their resistance to coagulation or flocculation. 

According to the resazurin test data, when incubating cells for 24 hours in a solution of nanoparticles, a pronounced dose-dependent effect was recorded in the form of inhibition of relative respiratory activity, with the following identified values of the minimum inhibitory concentration (MIC) that suppresses the growth of at least 50% of the studied strains (MIC50): 6.5 μg/mL for the reference strain and 10 μg/mL for the clinical strain (Figure 3).

Discussion

Currently, research in the field of green synthesis of nanoparticles using various biological objects is extremely relevant [12]. However, in most cases, biosynthesized nanoparticles are unstable, heterogeneous and are characterized by the irregular shape, which affects the possibility of their further use. As a result of our study, we managed to obtain AgNPs with a high degree of monodispersity and stability. According to UV-Vis spectroscopy, a peak with an absorption maximum at a wavelength of 470 nm was detected, which is consistent with the surface plasmon resonance of Ag°, occurring at a wavelength of 450–500 nm [13]. To study the antimicrobial effect, we achieved the concentration of nanoparticles from 1.00 to 1.56 μg/mL by the method of two-fold serial dilutions. According to the resazurin test, at an AgNPs concentration of 12.5–25 μg/mL, we observed no metabolic activity of bacteria, which confirms the bactericidal effect of the obtained nanoparticles. The MIC and minimum bactericidal concentration (MBC) of the particles for the reference strain were 6.5 μg/mL and 12.5 μg/mL, respectively. For the clinical strain, they were10 μg/mL and 25 μg/mL, respectively. Our data are consistent with some studies that have demonstrated the effectiveness of using biosynthesized nanoparticles against bacteria [14, 15]. The difference in the effective concentration of biosynthesized nanoparticles is explained by the fact that differences in the physicochemical properties of nanoparticles (i.e., shape, size, and presence of surface fragments) have a significant effect on their antibacterial effect. According to published sources, the antibacterial properties of silver nanoparticles negatively correlate with their size, i.e., their effect decreases with an increase in the diameter of the particles [16]. It is assumed that particles less than 15 nm are capable of penetrating the bacterial wall and exerting antibacterial effect more effectively than particles 15–20 nm in size. Our results showed that AgNPs exhibit a powerful antibacterial effect against Pseudomonas aeruginosa at low concentrations, although the particle size ranges 6–35 nm. One of the main mechanisms of action of silver nanoparticles is the disruption of the bacterial cell wall integrity and an increase in the permeability of the cytoplasmic membrane, the induction of lipid peroxidation, oxidative damage to DNA and proteins, as well as the blocking of a cellular signal transmission. In addition, when exposed to aerobic conditions, silver ions can be released from the surface of the particles, thereby exerting a powerful antimicrobial effect [17]. 

Conclusion

In our study, AgNPs were obtained and characterized via the green synthesis method using the cell-free extract of A. brasilense SR80. The obtained particles exhibited pronounced concentration-dependent antimicrobial activity against clinical and reference strains of P. aeruginosa in vitro. Our results emphasize the possibility of using bioengineered nanoparticles as alternative antimicrobial drugs.

Author contributions. M.A. Kupryashina – data analysis and manuscript preparation; E.G. Ponomareva, I.A. Mamonova – experiments; T.A. Kulshan – conceptualization, manuscript editing. 

Conflict of interest. None declared by the authors. 

Funding. The study was supported by the grant of the Russian Science Foundation No. 23-24-00570, Technology of Organopollutant Biodegradation Using Composite Materials Based on Biogenic Silver Nanoparticles and Phenoloxidases of Nonpathogenic Bacteria.