Biological activities of optimized biosynthesized selenium nanoparticles using Proteus mirabilis PQ350419 alone or combined with chitosan and ampicillin against common multidrug-resistant bacteria

Abstract

Background: One of the most common issues in the world is bacterial resistance and biofilms, which can prolong the healing period and the need for self-medication. Additionally, they may be linked to unsuccessful therapies, which raises death rates, healthcare expenses, and the need for additional hospitalization. Therefore, to protect the environment and improve human health, there is a need for the creative synthesis of novel antibacterial materials. Proteus mirabilis strain PQ350419 was isolated, identified, and utilized as an efficient bio-nano-factory for biosynthesizing selenium nanoparticles (Se NPs) and optimizing procedures. This study showcases a simple and cost-effective approach for green-synthesizing a selenium/chitosan/ampicillin nanocomposite (Se/CS/AMP) as a novel antibacterial and antibiofilm agent. Several analyses, such as transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, zeta analysis, and ultraviolet-visible (UV-Vis) spectroscopy, were utilized to confirm and characterize the production of Se NPs and Se/CS/AMP. The absorption peaks for Se NPs and Se/CS/AMP were identified to be between 350 and 360 nm. The XRD data revealed the crystalline composition of the Se NPs loaded with CS and AMP. The FTIR spectra confirmed the presence of proteins that act as supporting and binding agents during synthesis. The stability of the prepared nanomaterials is improved by a strong negative surface charge of - 24.27 mV for Se NPs and - 23.92 mV for Se/CS/AMP. The particle sizes of Se NPs and Se/CS/AMP are shown by TEM to be in the ranges of 88-98 nm and 86-129 nm, respectively. Se NPs, either alone or in combination with chitosan (CS) and ampicillin (AMP), exhibited strong antibacterial activity against methicillin-resistant Staphylococcus aureus ATCC 43,300, Bacillus cereus ATCC 14,579, Klebsiella pneumoniae ATCC 11,296, and P. mirabilis PQ350419 in a dose-dependent manner. Compared to Se NPs and the common antibiotic AMP, the Se/CS/AMP combination demonstrated superior antibacterial activity. In comparison to Se NPs (40, 70, 110, and 150 µg/ml, respectively), the nanocomposite produced MIC values of 30, 40, 60, and 100 µg/ml against B. cereus, S. aureus, K. pneumoniae, and P. mirabilis. When compared to untreated cells, treated cells exhibited significant morphological changes and deformities, such as cell wall distortion, the separation of the cell wall from the plasma membrane, the formation of vacuoles, and complete cell lysis, according to TEM ultrastructure studies of bacteria treated with nanocomposite. Se/CS/AMP at 100 µg/ml was sufficient to prevent biofilm formation by up to 50% in S. aureus, K. pneumoniae, and P. mirabilis. The cell viability of the Vero cell line was significantly reduced (p˂0.05) in the cytotoxicity test of Se NPs alone at a concentration of 40.95 ± 2.34 µg/ml, and in its nanocomposite at a concentration of 199.09 ± 2.61 µg/ml. This indicates the nanocomposite's safety by showing its minimal harmful impact on the Vero cell line.

Conclusion: Se/CS/AMP has revealed an antibacterial and antibiofilm agent that could be useful in various industrial, medicinal, and environmental applications. This study introduces a work that presents an alternative, safe, promising, and efficient nanocomposite for treating harmful bacteria in humans and animals. This treatment is based on the synergistic effectiveness of Se NPs, CS, and AMP.

Fig. 1

P. mirabilis isolates biofilm formation. * indicates considerably higher biofilms than those under control conditions (P < 0.05)

Fig. 2

UV–Vis spectroscopy and change in color studies during the formation of Se NPs using P. mirabilis isolates. (B) The reaction mixture at the beginning of the Se NPs production

Fig. 3

P. mirabilis AUF1 bacterial strain’s 16S rRNA sequences were used to create the phylogenetic tree. A bootstrap of 100 replicates was used to determine the number of branch nodes. Bootstrap values greater than 50% are shown. The strain of the genus Proteus was employed as an out-group

Fig. 4

Optimization of Se NPs production using P. mirabilis PQ350419. (A) Concentrations (5–10 mM) of Na₂SeO₃. (B) Effect of different mixing ratio between cell-free bacterial metabolites and Na₂SeO₃ (1:1–1:10 v/v%). (C) Different incubation periods through Se NPs biosynthesis. (D) Effect of temperature (10–50 °C). (E) Effect of different pH value on Se NPs formation

Fig. 5

FTIR spectra of cell-free P. mirabilis PQ350419 metabolites, CS, Se NPs, and Se/CS/AMP

Fig. 6

XRD patterns of Se NPs, and Se/CS/AMP

Fig. 7

TEM micrographs of Se NPs; (A), and Se/CS/AMP; (B). Bar scales = 200 nm

Fig. 8

Zeta potential of Se NPs; (A), and Se/CS/AMP; (B)

Fig. 9

Standard curve of AMP in pure water

Fig. 10

Antibacterial activity of Se NPs and Se/CS/AMP, CS, and AMP using agar well diffusion method against B. cereus ATCC 14,579, methicillin-resistant S. aureus ATCC 43,300, K. pneumoniae ATCC 11,296, and P. mirabilis PQ350419

Fig. 11

Agar well diffusion method test of Se NPs and Se/CS/AMP against B. cereus ATCC 14,579; (A), methicillin-resistant S. aureus ATCC 43,300; (B), K. pneumoniae ATCC 11,296; (C), and P. mirabilis PQ350419; (D), in comparison to AMP and CS

Fig. 12

Minimum inhibition concentration of Se NPs; (A), and Se/CS/AMP; (B), against the tested bacteria

Fig. 13

Minimum bactericidal concentration of Se NPs and Se/CS/AMP against the tested bacteria

Fig. 14

TEM micrographs showing the antibacterial action of Se NPs; (B), compared to Se/CS/AMP; (C) against P. mirabilis PQ350419. Untreated bacterial cells; (A), displayed normal cell structures. Treated P. mirabilis PQ350419 cells; (B&C), displayed separation between the cell wall (CW) and cytoplasmic membrane (PM); (white arrowhead), vacuole formation; (V), complete cell lysis; (CL), disintegrated cytoplasm (DC), and sever malformation and distortion; (yellow arrows)

Fig. 15

Antibiofilm potential of Se NPs and Se/CS/AMP in different concentrations (50, 100, and 150 µg/ml) against biofilm-producing bacteria (S. aureus ATCC 43300, K. pneumoniae ATCC 11296, and P. mirabilis PQ350419)

Fig. 16

The cytotoxic effects of Se NPs and Se/CS/AMP against Vero cell line