Evaluation of the Antibiofilm and Antimicrobial Effects of Selenium Nanoparticles on Multidrug-Resistant Salmonella Isolates

Introduction

Salmonella is a rod-shaped, facultative anaerobic, non-spore-forming, Gram-negative bacterium that can cause disease among humans as well as animals. The bacteria are a member of the Enterobacteriaceae family.¹

The ongoing face-off between the multidrug resistance (MDR) of Salmonella on one hand and the drug development and use of antibiotics and other antipathogenic substances on the other hand remains an important global problem.² The infectivity of these bacteria and the threats that follow to many industries, including healthcare, water, food, and energy, wherein Salmonella could cause a terrible deal of harm, continue to be highlighted among critical world concerns.³ The increasing drug resistance among Salmonella species is an emerging problem and the syndrome of MDR keeps getting worse. Given Salmonella’s massive toll on public health, ongoing alerts for its drug resistance pattern will be needed to understand resistive evolution, enable proper clinical treatment protocols, and curb its transmission and infection.⁴

The past decade has witnessed a great increase in the prevalence of multidrug-resistant Salmonella species, with MDR Salmonella typhimurium imposing such significant therapeutic challenges because of rising morbidity and mortality rates sustained by its infection. Alarmingly, accession of mortality rates is by the MDR-ACSSuT resistance pattern that is 4.8 times higher than that for non-MDR-ACSSuT strains, with resistance including Ampicillin, Chloramphenicol, Streptomycin, Sulfamethoxazole, and Tetracycline.⁵ Beyond cellular drug resistance mechanisms, S. typhimurium acquires adaptive resistance mechanisms, such as the capability to form biofilms. Observations of biofilm formation are postulated to be aiding in significantly increasing the drug resistance in the strains.⁶ In addition, studies have increasingly shown that virulent multidrug-resistant bacterial pathogens are emerging from a variety of sources, thereby stressing the need for proper use of antibiotics. This is coupled with the pressing imperative for the design and implementation of rapid, reliable diagnostic tools for the detection of emerging virulent multidrug-resistant strains.^7–9

The increased pathogenicity of Salmonella is an aggravating factor increasing public health consequences.¹⁰ Biofilm formation by Salmonella species on biotic or abiotic surfaces is a well-documented mode of bacterial existence.^11,12 Biofilm formation is considered clinically relevant since about 80% of chronic bacterial infections are associated with this mode of growth.^13,14 Biofilms can increase bacterial survival rates by promoting resistance against antimicrobial agents and decoying immune defenses, thereby allowing them to play an important role in chronic and device-related infections.^15–17

A biofilm is a membrane-like structure composed of bacteria encapsulated by extra-cellular macromolecules forming a hydrated matrix during bacterial growth. Research has established that almost all Salmonella strains can form biofilms, and most have been shown to be highly proficient in this.¹⁸ The formation of biofilms greatly increases bacterial resistance to stress conditions, which include drying, extreme temperature, antimicrobial agents, and disinfectants, and hence facilitates the survival of Salmonella in diverse environments.¹⁹ In-depth investigation of biofilm formation mechanisms could aid in developing preventive measures against Salmonella spread and infection.²⁰

Therefore, further studies are needed for a more effective Salmonella biofilm control. The present study will focus on drug resistance to Salmonella species and will test those in vitro for biofilm formation in relation to the gene expression of biofilm-producing strains, which will provide important data that can be utilized for the clinical treatment of Salmonella infections. The context explored in connection with the study involved the observation of SeNPs as a possible candidate to hinder biofilm-associated genes in Salmonella.

The unique properties of metal NPs have led to their great interest as antimicrobial agents. Silver (Ag), Zinc oxide (ZnO), Titanium dioxide (TiO₂), Copper (Cu), Iron oxide (Fe₃O₄), Selenium (Se), and Gold (Au) metal NPs have been widely studied for their antimicrobial properties.²¹ Previous studies have suggested that NPs can operate by mechanisms other than those used by conventional antibiotics, thus perhaps improving their activity against MDR bacteria. Antibacterial studies have shown that these NPs penetrate bacterial cells, bind to receptors on their surface, and exert their action by inflicting intracellular damage to finally kill the cells. NPs are also able to inhibit essential metabolic enzymes responsible for bacterial proliferation and respiration. Furthermore, some of these NPs can interfere with biofilm formation by disrupting the signaling pathways involved, preventing infections associated with biofilm.^22,23 This review presents the potential of metal and metal oxide NPs as some of the most powerful therapeutics to treat biofilm, specifically focusing on their inhibition of the bacterial biofilm-associated genes.

Materials and methods

Results

Characterization of Selenium Nanoparticles (SeNPs)

SEM images of biosynthesized selenium nanoparticles at magnifications of 5,000×, 10,000× and 20,000× with an accelerating voltage of 20 kV show nanomaterials sparsely aggregated in certain areas, but even distribution across most of the surface. At higher magnifications (10,000× and 20,000×), aggregation is reduced, allowing for uniform distribution of particles, as shown in Figure 1. The overall particle size is within nanometer range, proving them appropriate for biological purposes.

Figure 1. Morphological characterization of biosynthesized SeNPs using SEM.

Scanning electron microscopy (SEM) image illustrating the surface morphology of biosynthesized selenium nanoparticles (SeNPs), acquired at an accelerating voltage of 20 kV and a magnification of 10,000×. The scale bar represents 5 μm.

Biofilm formation profile of bacterial isolates

The ability of the bacterial isolates to form biofilms was determined using the Congo Red and Microtiter Plate (MTP) assays. In the Congo Red assay, all 32 isolates were identified as biofilm producers, showing 100% positivity for biofilm generation. In the Microtiter Plate assay, 24 isolates (75%) were classified as strong biofilm formers, 7 isolates (22%) as moderate biofilm formers, and 1 isolate (3.1%) as a weak biofilm former, as summarized in Table 4, Figure 2.

Table 4. Biofilm formation profile of Salmonella isolates (n = 32).

Assay	Category	Number of isolates	Percentage (%)
Congo Red	Producer	32	100
Microtiter Plate	Strong	24	75
	Moderate	7	22
	Low	1	3.1

Figure 2. Biofilm formation profile of bacterial isolates assessed by Congo Red and Microtiter Plate assays.

Biofilm formation profile of the bacterial isolates as assessed by the Congo red agar method and the microtiter plate assay, showing the distribution of isolates classified as strong, moderate, or low biofilm producers.

Dose-dependent downregulation of csgD and gapA gene expression revealed by RT-qPCR

Relative expression levels of the csgD and ssrB genes were determined by RT-qPCR, with gapA as the housekeeping gene for normalization. Fold change values (2^-ΔΔCt) were calculated relative to the control group and are shown as mean ± SD.

For csgD, the control group showed a stable expression (1.01 ± 0.18). Treatment at 12.5% concentration decreased expression (0.16 ± 0.01), whereas further decreases were recorded at increased concentrations: 0.09 ± 0.06 at 25%, 0.08 ± 0.04 at 50%, and 0.03 ± 0.02 at 100%, as shown in Figure 3.

Figure 3. Dose-dependent downregulation of csgD gene expression in Salmonella isolates treated with selenium nanoparticles (SeNPs).

Gene expression levels were quantified using the 2^−ΔΔCt method and normalized to the housekeeping gene gapA. Data are presented as mean ± SD.

For ssrB, the control group showed stable expression (1.02 ± 0.23). At 12.5% concentration, moderate reduction was detected (0.91 ± 0.29), followed by stronger decreases being recorded at 25% (0.75 ± 0.30), 50% (0.56 ± 0.23), and 100% (0.28 ± 0.16), as shown in Figure 4.The RT-qPCR results thus show that both csgD and ssrB gene expression was downregulated in a dose-dependent manner with respect to the treatment, with stronger reduction seen at higher concentrations.

Figure 4. Dose-dependent downregulation of ssrB gene expression in Salmonella isolates treated with selenium nanoparticles (SeNPs).

Relative gene expression was calculated using the 2^−ΔΔCt method and normalized to the housekeeping gene gapA. Data are presented as mean ± SD.

Discussion

The above factors, among others, should contribute to the improved incidence of salmonellosis in persons less than 20 years old. These factors include lower doses needed to infect children, more symptomatic infection likely, more biological samples could be obtained for laboratory analysis, and heightened hospitalization rates to document better the cases of infection.³³ Gender-specific analysis demonstrated that infection rates were higher among females under 20 than among their male counterparts. This trend follows a previous study that reported that women are more exposed to contaminated food during preparation, especially raw meat and eggs.³³ Similar national indices were found internationally. For example, in Hong Kong (2003–2011), the ratio for females to males was 1.24:1; in the USA (2011), it was 1.09:1. Several studies conducted in London (2007–2011) and in Ireland (2012) reported negligible gender differences with ratios set at 1:1 and 0.90:1.11, respectively.^34–36 Moreover, Green (1992) had indicated that generally, females aged between 15 and 44 years recorded the incidence rates of enteric infections, including salmonellosis, higher than that for males.³⁷

Bacterial resistance has emerged as an issue acknowledged by the global community as a health hazard. Multi-drug resistant bacterial pathogens are rampant throughout the world. While the greatest numbers of pathogenic strains with serious resistance to drugs are those resistant to broad-spectrum cephalosporins and fluoroquinolones, Salmonella is one of them.³⁸ Clinical studies assess the strains resistant in general to common antibiotics such as ampicillin, nalidixic acid, and streptomycin, cefoperazone, and tetracycline.³⁹ The latest research has highlighted the serious health implications of multidrug-resistant Salmonella infection. For example, individuals infected with Salmonella resistant to one or more clinically important antibiotics are three times more susceptible to bloodstream invasion, and require hospitalization, compared with patients infected with the susceptible strains.⁴⁰ Excessive antibiotic uses have enabled the bacterial resistance-rising phenomenon as well as the resistance-gene arrays.⁴¹

Bacterial biofilm (BBF) comprises a unique survival strategy that enables adaption of bacteria to environmental stress. Reportedly, most Salmonella strains can form biofilms that endow these bacteria with resistance from bactericidal actions such as host antibodies and often cause refractory infections. Biofilms are organized microcolonies differentiated by a complex communication system known as bacterial quorum sensing. Latest studies have portrayed quorum sensing as a major regulator in the control of biofilm formation, development, and functionality.⁴²

Salmonella virulence inside the host is mainly determined by its ability to form biofilms.³⁴ Biofilms provide an adaptive response to environmental stresses and antibiotics by altering bacterial gene expression for further resistance against both⁴³). Those feature characteristics by which Salmonella biofilms develop include causative factors composed mainly of curli fimbriae and cellulose in those aggregates.⁴⁴ Biofilm formation is mainly regulated by the curli subunit gene csgD, belonging to the LuxR family.⁴⁵ Post-transcriptional modulation is available through various environmental signals, including those from transcription factors like c-di-GMP and small RNAs (sRNA) that can act on csgD expression.⁴⁶

In the current study, SeNPs have a reducing effect on the Salmonella gene expression due to their anti-bacterial properties including Cathodestrupture of the cell wall; Referring DNA and protein interactions, Anti-biofilm development, Production of reactive oxygen species (ROS).⁴⁷ Nanotechnology gives a new boon to biofilms because engineering materials at the atomic and molecular levels provides high surface-to-volume ratios and reactivity.⁴⁸ Nanoscale metals can therefore change their physicochemical properties and new abilities that allow them to penetrate and inhibit biofilm formations.⁴⁹

Three stages characterize the interaction between nanoparticles and biofilms: transport near the biofilm, adherence to the biofilm surface, and migration within the biofilm. These processes differ by environmental conditions and composition of the extracellular polymeric matrix that hosts some nanoparticle property.⁵⁰ Some nanoparticles have intrinsic properties of anti-biofilm activity because they contain several antibacterial components, e.g. metal oxides or cationic surfactants.⁴² Their large surface area combined with high reactivity and surface functionalization leads to an excellent-wave efficacy in biofilm destruction. Besides, nanoparticles also act as quorum-quenching)QQ) agents that interfere in bacterial cell-cell communication or inhibit quorum-sensing signaling that in turn forms complexes with signaling molecules and receptors.⁵¹

This establishes selenium nanoparticles as a very promising antibiofilm and antimicrobial agent against multidrug-resistant Salmonella with probable use in clinical and food safety applications.

Conclusion

Under dose-dependent conditions, selenium nanoparticles significantly reduced biofilm-related gene expression (csgD and ssrB) and strongly inhibited the growth of multidrug-resistant Salmonella. Therefore, SeNPs may be contemplated as a potential adjunctive treatment for Salmonella infections.

Ethical considerations

The Ethics Committee of the College of Science, Al-Mustansiriyah University approved this study on 2 January 2024 (Ref. No. BCSMU/1724/00073 M). The study involved the collection of clinical isolates and human blood samples. Written informed consent was obtained from all adult participants prior to sample collection. For participants under 18 years of age, written informed consent was obtained from their parents or legal guardians, in accordance with the Declaration of Helsinki and internationally accepted ethical standards for biomedical research. Consent for publication was obtained from all participants (or their legal guardians, where applicable), and all data were handled anonymously to ensure confidentiality.