Advances in silver nanoparticles: a comprehensive review on their potential as antimicrobial agents and their mechanisms of action elucidated by proteomics

1Nanomaterials are structures with dimensions between 1 and 100 nanometers, encompassing the field of nanotechnology. They include nanostructures, nanocomposites, nanofibers, and nanofilms, and can be composed of polymers, biomaterials, ceramics, metals, among others. They encompass a variety of structures with different morphologies, such as rods, tubes, films, and particles. Nanoparticles, a subset of nanomaterials, are specifically characterized by their three-dimensional nanoscale size and generally have spherical or quasi-spherical shapes. Their unique properties, such as high reactivity and large surface area, make them particularly suitable for applications in drug delivery, imaging, and catalysis.

2Obtaining nanoparticles can be carried out by chemical, physical and biological methods, often involving high costs and the use of toxic substances. On the other hand, synthesis using natural substances, such as phytochemicals, has been widely discussed due to their reducing potential, biocompatibility and low environmental toxicity. Green nanotechnology, based on the principles of green chemistry, seeks economic, social, health and environmental benefits by prioritizing the reduction of the generation of hazardous chemical waste and promoting safer applications.

3Analytical techniques for the physicochemical characterization of nanoparticles are fundamental for the detailed investigation of their properties, including structure, crystallinity, morphology, size, and surface chemistry. UV–Visible spectroscopy is used for the primary characterization of colloidal suspension particles, confirming the formation of NPs by the intensity of the surface plasmon resonance (SPR) band. The Dynamic Light Scattering (DLS) technique allows the determination of the hydrodynamic particle size, influenced by Brownian motion as a function of the diffusion coefficient, analyzing fluctuations in the intensity of scattered light to determine particle diffusion and directly relate it to size. The relationship between the diffusion coefficient and size is variable for spherical particles, as the diffusion coefficient decreases as the hydrodynamic radius of the particles increases, as described by the Stokes-Einstein equation. Additionally, it provides the polydispersity index, indicating system homogeneity. Nanoparticle Tracking Analysis (NTA) individually tracks the movement of each particle, determining its size based on Brownian motion. The Zeta potential measures the surface charge and stability of colloidal suspensions, indicating the tendency of particles to aggregate or repel each other. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is used for multielemental investigation, quantifying silver uptake in different systems. X-ray Diffraction (XRD) provides information on the crystallinity level and molecular structure of nanoparticles. Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectroscopy identify the surface chemical composition and investigate particle functionalization, vibrational, and rotational modes. X-ray Photoelectron Spectroscopy (XPS) analyzes the surface chemistry of NPs, while Energy Dispersive X-ray Spectroscopy (EDX) identifies and quantifies the elemental composition of materials. Nuclear Magnetic Resonance (NMR) spectroscopy is used to identify and quantify surface functional groups. Imaging techniques, such as Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), provide detailed images of particle morphology and size, allowing the creation of size distribution histograms through statistical analysis. Atomic Force Microscopy (AFM) allows for the analysis of aggregation state, dispersion, topography, structure, shape, and size of nanoparticles. Together, these techniques provide comprehensive data on the structural, chemical, and morphological properties of nanoparticles, enabling an in-depth investigation of their characteristics.

4Silver has a long history as an antimicrobial agent and has been used since ancient times, with documented evidence in several medicinal applications. In 1881, Credé’s method introduced the use of a solution containing 2% AgNO3 to treat neonatal conjunctivitis. In the 19th century, doctors used silver thread to suture surgical wounds, while during the First World War, silver foil was applied to soldiers’ wounds to prevent infection and aid wound healing. By the early 20th century, colloidal silver became popular in hospitals as a germicidal agent. Additionally, silver salts were used to treat various infections, such as conjunctivitis, gastroenteritis, gonorrhea and syphilis.

5Silver nanoparticles (AgNPs) are widely described in the literature as having a broad biocidal spectrum, covering Gram-positive and Gram-negative bacteria, fungi, viruses and mycobacteria, in addition to acting synergistically with antibiotics to enhance their effectiveness. These properties are based on diverse and complex mechanisms, including the generation of reactive oxygen species (ROS), DNA damage, impairment of bacterial cell membranes and inhibition of protein synthesis.

6Recent studies have highlighted the effectiveness of AgNPs against a variety of bacterial strains, including clinical isolates and standard strains (ATCCs, NCTCs, PCCs, MTCCs) of various species, such as Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhi, Enterococcus faecalis, Enterobacter spp., Bacillus subtilis, Micrococcus luteus, Proteus vulgari, among others. Furthermore, the synergy between AgNPs and antibiotics, such as penicillin G, amoxicillin, erythromycin, clindamycin, vancomycin, piperacillin, ciprofloxacin, tetracycline, levofloxacin, cefixime, cefazolin, neomycin, rifampicin, gentamicin, chloramphenicol, imipenem; has been shown to increase the effectiveness of these medications.

7This review addresses the mechanisms of action of AgNPs against planktonic and biofilm bacteria at the proteomic level, as well as their biomedical applications, aiming to enhance formulations and investigate synergism with antibiotics, highlighting advances relevant to nanomedicine and public health.

8The diverse applications and the increase of AgNPs in the marketplace makes them inevitably present in the environment, as they are released not only during the use of products such as cosmetics, wound dressings, textiles, food packaging and personal care products, but also through their productive processes. This fact entails the ingestion of this NPs by humans by food or drinks that were in direct contact with these NPs or through contaminated by water containing these nanostructures, and their intake can also occur via dermal contact, inhalation or intravenous or intraperitoneal injections.

9Once inside the body AgNPs are distributed through the organs and tissues, firstly accumulating in the lungs when entering via the respiratory tract. These nanoparticles can penetrate both healthy or injured skin when present in formulations for topical application such as cosmetics and wound dressings or even textiles and then enter circulatory system. Toxicity and pathological symptoms of AgNPs intake are modulated by some properties of these nanostructures, such as size, stability and aggregation condition. summarized their toxic effects on different animal models, with different organs being affected.

10administered commercial AgNPs (15–30 nm) coated with polyoxyethylene glycerol trioleate and Tween 20 (polyoxyethylene (20) sorbitan monolaurate) to rats via oral or intravenous injection and after 24 h measured their content on different tissues and feces. For the orally administered group, AgNPs accumulated in feces and colon, while intravenous administration resulted in a more distributed accumulation, being present mainly in lungs, then in feces, followed by the colon, intestine, kidney, testis and thymus, with an expected silver ions content below 15%. still also indicate the reduction in AgNPs size to 5 to 12 nm and the formation of silver acetate NPs of 6 nm in animals treated with silver ions (AgNO3).

11Since the gastrointestinal tract is one of the main entry ways for many invaders, as pathogens and toxins, including AgNPs, evaluated the intestinal toxicity of these NPs in vivo. Mice were exposed daily for 4 weeks with 10 mL/kg body weight of commercial suspensions of AgNPs capped with poly(vinylpyrrolidone) of 5 mg/mL through oral gavage were compared to animals being treated with other NPs or water. The authors indicated severe damage for the animals treated with AgNPs including damage in the epithelia structure, reduction in the mucosa layer thickness and alterations in the local microbiota. Additionally, the interaction of immune cells with these NPs resulted in the generation of reactive oxygen species (ROS) that induced uncontrolled apoptosis of dendritic cells, affecting the normality of the immune microenvironment in the intestine of these animals. Among the tested NPs (Ag, TiO2, SiO2, and ZnO) silver was the most harmful.

12The cytotoxicity of AgNPs is generally related to the production of large amounts of ROS, affecting cytoskeleton, DNA, DNA repair enzymes and inducing cell apoptosis. However, these effects are associated with size and the synthetic methods employed. Larger NPs present less cytotoxicity due to the reduction in surface area and, consequently, the reduction in interaction with cell membranes and organelles. biosynthesized AgNPs employing aqueous extract of a mushroom (Agaricus bisporus) and evaluated their cytotoxicity against human skin fibroblasts, obtaining cell viability of 98.2% for the diluted solutions (0.04 μg/mL) until 82.3% for 25.0 μg/mL samples. Besides the dose-dependent cytotoxicity, it is worth pointing out that the size of the NPs was 766 nm, and the use of safer aqueous extracts as reducing agents instead of commonly applied chemicals for this purpose. This approach eliminates purification steps to reduce the presence of these compounds that tend to increase cytotoxicity.

13compared AgNPs prepared by chemical (sodium borohydride) and green (clove bud extract) methods of synthesis and observed a better larvicidal (Aedes aegypti) effect with lower toxicity (Daphnia magna) for the samples prepared with plant extract. Despite the recognized toxicity of AgNPs some approaches can reduce this drawback, such as the use of natural reducing and capping agents, the control of size and the use of these NPs in combination to other antimicrobial agents to attempt microbial control through different and complementary mechanisms, reducing the necessary concentration of AgNPs.

14Nanomaterials are structures that exist on the nanometer scale (10-9). According to the American Society for Testing and Materials (ASTM), nanoparticles are a specific category of particles with lengths in two or more dimensions within the range of 1 to 100 nm.

15The characteristics of these metallic nanoparticles depend on factors such as size, morphology, stability, surface charge, and other physical and chemical properties. These properties can be influenced through the kinetic interaction conditions between metal ions and reducing agents, by modifying synthesis methods, characteristics of the reducing agents, and the use of stabilizers. Additionally, various morphologies of AgNPs can be obtained, such as cubic shapes, spherical, nanorods, nanowires, nanobars, triangular and pyramidal, in a controlled manner by modifying thermodynamics and kinetics during synthesis, through factors such as the type of reducer (chemical or physical), use of solvent systems, variations in temperature, pH, pressure, and controlled atmosphere.

16The preference for biological methods in the synthesis of AgNPs has driven significant advances in the search for more ecological and efficient processes. This approach is based on the use of bacteria, plant extracts, and biomolecules as non-toxic reducing agents, offering a safe, environmentally friendly, and economically advantageous alternative for AgNPs production. Biological methods are distinguished by their safer and less harmful approach, supported by green nanotechnology stemming from ‘green chemistry’, aiming for beneficial innovations for health, the environment, and industry, ensuring efficiency and safety in meeting current needs with greater sustainability.

17AgNPs can be used in different sectors, such as medicine, engineering, agriculture, and the environment sciences. Major applications include surface coating and medical device coating, advanced dressings, theranostics, cosmetics, textiles and electronics, sensors, and catalysts. Their properties include antibacterial, antifungal, antiviral, anti-inflammatory, anti-angiogenic, and antitumor activities.

18Bacterial resistance to antibiotics represents a global health challenge. The World Health Organization (WHO) warns that drug-resistant pathogens significantly contribute to high mortality rates. Consequently, research and development of new antimicrobial agents have attracted considerable interest. Among the various known metallic nanoparticles, such as Au, Pd, Cu, and Zn, silver stands out as one of the most effective in combating pathogenic microorganisms. Studies often cite its broad-spectrum antimicrobial capacity due to its large surface area and multivalent interactions.

19Table 1 presents significant studies exploring the potential of AgNPs in various applications, such as antimicrobial, antifungal, and antiviral action. These studies address synthetic methods, morphology, size, and the different targets to which these nanoparticles are currently being applied.

20The search for new antimicrobial agents has been driven by the increasing number of infections presenting bacterial resistance to conventional antibiotic treatment, a which poses significant challenge for global health. The indiscriminate use of antibiotics has led to the development of multidrug-resistant microorganisms and consequently antimicrobial resistance (MDR;; ). This microbial resistance represents a serious health problem, potentially raising morbidity and mortality rates, especially in pandemic and epidemic diseases, as highlighted by the World Health Organization (WHO). The emergence of bacteria resistant to antibacterial agents underscores the need to development of more effective antimicrobial agents to overcome these resistance profiles.

21According to the WHO, pathogens identified as top-level are categorized as ESKAPE, namely: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. These pathogens pose significant challenges to human health, as they are the most prevalent in infections caused by MDR. Gram-negative bacteria exhibit a more pronounced profile of microbial resistance compared to Gram-positive strains.

22The use of nanoparticles represents a substantial advance in the development of new therapeutics to tackle microbial resistance. Several authors have demonstrated the ability of AgNPs to significantly reduce biofilm formation and bacterial adhesion. There is also the possibility of employing NPs in synergy with traditional antibiotics. Due to the unique properties of these nanomaterials, they could potentially reduce the dose and toxicity of antibiotics optimizing treatment compared to the exclusive use of antibiotic.

23The advantages of using AgNPs lie in their ability to interact with different bacterial mechanisms by targeting multiple pathways, thereby potentially increasing the spectrum of antimicrobial action. By interacting with a broad spectrum, they can interfere with metabolic pathways by inducing ROS formation, inhibiting and/or modifying enzymes and proteins, reducing cell permeability, and causing homeostatic imbalance.

24Several authors have demonstrated the importance of synergy between silver nanoparticles and antibiotics, as evidenced in Table 2. This table presents relevant information on the interaction of these NPs with different antibiotics, highlighting potential synergistic effects that can result in greater effectiveness in combating various strains of microorganisms, including resistant ones.

25In summary, the diverse applications of AgNPs as antimicrobial agents encompass a wide range of microorganisms of significant interest in hospital setting, particularly for hospital-acquired infections. By exploring these applications, it is possible to advance the search for innovative solutions to combat and reduce microbial infections and MDR strains through the use of nanotechnology and the unique properties of silver. Current applications of nanomedicine demonstrate significant advancements in the field. These technologies improve existing medical products, offering substantial benefits for patients and healthcare professionals in terms of efficacy, safety and infection control.

26Bacteria can be found in a mobile, also called planktonic state, being free to disperse and go in search of the nutrients present in their microenvironment or attached to surfaces in a community of microorganisms, called biofilm. In biofilms, bacteria can be protected against harmful conditions, as antibiotic agents, by an extracellular polymeric matrix composed of polysaccharides, protein, lipid and extracellular DNA, produced to promote their adhesion. This makes it difficult for antimicrobial substances to penetrate through the biofilms, making these multicellular structures an additional challenge to overcome microbial contamination (Figure 1). Surgical site infections are common examples of biofilms infections occasioned by surgery procedures that can increase the recovery time, medical costs or even result in death.

27Besides the increased defense obtained with this polymeric matrix, that can act as a semipermeable barrier, some phenotype properties of these microorganisms are affected by the formation of these structures, such as the growth rate and gene transcription, which are commonly increased, allowing the formation of biofilms within a few hours. Furthermore, the bacterial genetic diversity inside the biofilm is mediated by the communication between the cells close to the surface (in contact to the outer environment) and the inner cells through the exchange of plasmids, which strongly contributes to the development of antimicrobial resistance.

28Although biofilms are statically attached to inert or living surfaces, when there is a reduction in the nutrients supply or other changes in physical or chemical factors, the biofilms can propagate by the detachment of clumps of cells or individual organisms, in a process known as seeding dispersal, allowing the proliferation of the infection. Due to all these differences between planktonic and static multicellular structures, biofilm infections may require different agents or approaches for treatment, hence the need for the development of antibiofilm agents.

29Among the established options to control biofilms formation, we can include ultraviolet irradiation, chlorine and chloramine treatment, hydrogen peroxide and nitrous oxide. However, in some situations these agents cannot be used because of their toxicity, other potential damages or even due to the antibiotic resistance. The environment of biofilms presents a gradient of oxygen content, decreasing from the surface to its deep layers due to the oxygen consumption by the first bacteria, forming anaerobic niches. Additionally, differences in pH between the different regions of these structures can be noticed, resulting in a variety of cells that may differ genotypically and phenotypically. Since higher levels of conventional antimicrobials are required to combat biofilm infections, the risk of systemic toxicity also rises. Nanoscale antimicrobial agents emerge as a promising solution, aiming to enhance antibiofilm activity without inducing harmful side effects (.; ).

30When applying metallic nanomaterials to control biofilms growth, three critical steps are known to govern their action; (i) the interactions with the biofilm surface, affected by NPs charge; (ii) entry in the biofilm, dependent on nanomaterials properties such as surface chemistry and charge, size and concentration, as well as to biofilm surface composition and maturity; and (iii) the ability of NPs and their ions to migrate internally to interact with all the components of the biofilm.

31AgNPs have been extensively studied as antibiofilm agents, even being used commercially as a coating material to prevent the infection of surfaces of medical supplies as catheters and wound dressings. Table 3 presents the results of some applications of AgNPs as antibiofilm agents.

32Table 3 presents a variety of synthesis methods applied in the production of AgNPs with antibiofilm properties. Traditional chemical reduction procedures typically involve hazardous and sometimes expensive substances, necessitating purification steps to remove excess unreacted reagents and reduce toxicity. This increases both the time and cost of nanoparticle production. In contrast, green synthetic methods not only make the produced nanoparticles safer and more cost-effective but also environmentally friendly and conducive to scalability. These methods utilize natural compounds such as proteins, reducing carbohydrates, polyphenols, flavonoids, terpenes, and other secondary metabolites to reduce metal ions and/or act as capping agents. Two prominent classes of green synthesis for producing antibiofilm AgNPs have emerged: biosynthesis and phytosynthesis. Biosynthesis involves algae, microorganisms, or their cell-free extracts (filtrates or supernatants), while phytosynthesis utilizes plant extracts.

33Aiming to develop a sustainable synthesis method for AgNPs, applied a hydroethanolic solution of grape canes, a waste product of vine growing, to sustainably produce mono or polydisperse NPs suspensions that were effective against biofilms, also revealing a synergy between the AgNPs and the plant extract.

34used microalgae, brown seaweeds, to biosynthesize AgNPs that presented better action against biofilms than the reference antibiotics kanamycin and ampicillin, while and applied biological compounds (bacterial or fungal metabolites) from the supernatant or biomass of microorganisms as reducing, stabilizer, and dispersant agents, both obtaining AgNPs with good results in biofilm control.

35Physical methods can also be applied in AgNPs production. This class of synthesis methods can produce NPs with minimal impurities and, sometimes, in a most effective way than chemical and biological methods. One physical method applied for antibiofilm AgNPs production is the laser ablation in liquid, a low-cost and eco-friendly procedure in which a metal foil (or more than one, when producing multi element metallic NPs) is immersed in a solvent, followed by ablation with a laser beam, which results in NPs free of organic contaminants or capping agents, needing only of purification steps to change the solvent, for example, from a hydroethanolic solution to water.

36Electrical arc discharge (EAD) is another example of this class in which two electrodes are positioned inside an open vessel containing ultra-pure water and, with the initiation of the arc discharge the silver electrode vaporizes being then quenched by the cold solution, forming nanoparticles.

37Although assessed the antimicrobial activity of the AgNPs covered with carbon shells produced by EAD only against planktonic bacteria, the authors reported the inhibition of mrkA (biofilm forming gene), fimH (virulence adhesion gene) and rmpA (mucoid factor encoding gene) genetic expression in P. aeruginosa, affecting its intracellular signaling pathway., in turn, observed the importance of inhibiting the polysaccharide synthesis locus (psl) gene for P. aeruginosa biofilm formation control. The authors produced AgNPs and conjugated it with TSC, antimicrobial compound that did not present an expressive effect in gene inhibition, but enhanced the antibiofilm activity when conjugated with the NPs. Since Psl, along with Pel polysaccharide are responsible for the formation of the primary matrix of biofilm, this effect of partial inhibition can be understood.

38In view to interfere in quorum sensing (QS)-regulated P. aeruginosa biofilm formation, AgNPs were phytochemically prepared using aqueous extract of K. paniculata leaves, an anti-QS substance. The NPs reduced biofilm formation of a multidrug-resistant model strain of P. aeruginosa almost completely, downregulating several QS-virulence factors, such as pyocyanin, pyochelin, alginate, protease and rhamnolipid, and QS-regulated genes (lasR, rhlR, rhlI, lasI, lasA, lasB, rhlA, and rhlB; ).

39produced nanocomposites of AgNPs modified with antimicrobial peptides and polydopamine that were able to improve both HEK293T cells viability and antibacterial activity against S. aureus, E. coli, and P. aeruginosa. Thickness, biomass and the semi-quantitative analysis of crystal violet staining results indicated an effective reduction of biofilms of all strains, with the reduction of the mRNA expression of biofilm-related genes las I, rh II and fim H.

40The size of AgNPs significantly impacts their ability to combat biofilms. The dense extracellular matrix of biofilms acts as a barrier to NPs penetration. Moreover, smaller AgNPs possess a greater surface area, allowing for increased interaction with biofilms and potentially enhanced antibiofilm activity. As shown in Table 3, most antibiofilm AgNPs are under 100 nm, with many falling below 50 nm. Polydispersity also plays a role, as noted that polydisperse NPs were more effective than monodisperse suspensions of AgNPs in controlling C. albicans biofilms.

41compared the effectiveness of silver nitrate and AgNPs against S. aureus and P. aeruginosa biofilms, finding similar or lower MIC values for the phytosynthesized NPs. This suggests that silver ions, released from AgNPs play a crucial role in inhibiting microbial growth within biofilms, overcoming the compact biofilm structure that impedes antibiotic penetration.

42Many microorganisms developed resistant strains that are not susceptible to the classic antibiotics. In these cases, a common strategy is the combination of antimicrobial agents aiming to obtain a synergistic effect, since these components can present complementary mechanisms to inhibit microbial growth and form stable complexes via weak intermolecular interactions that prolong their release and reduce their potential local toxicity. This approach can include mixing AgNPs with classical antibiotics, plants extract rich in bioactive molecules as antioxidants or magnetic elements, which allows the use of magnetic fields to damage biofilms, promoting NPs penetration by highways created via mechanical disruption without additional associated risks.

43mixed AgNPs with Triclosan (5-chloro-2-(2,4-dichlorophenoxy) phenol), a synthetic antimicrobial compound used in a variety of consumer products, including hygiene products and to coat medical supplies as sutures (PDS Plus®, Vicryl Plus® and Monocryl 250 Plus®) or trans-cinnamaldehyde (TCA), a phytochemical extracted from cinnamon with antimicrobial activity and generally recognized as safe by U.S. Food and Drug Administration (FDA). For both combinations the AgNPs effect against S. aureus and E. coli biofilms formed in surgical sutures was improved, with additive effect when combining the NPs to Triclosan and synergistic effect when used with TCA.

44Besides combining substances, an approach applied in biofilms control involves the use of hyperthermia, i.e., the use of heat as a therapeutic method to control microbial growth. Hyperthermia is specially used in cancer treatment and involves cell death by local heating (41°C to 50°C). observed important biofilm reduction combining AgNPs functionalized with mercaptopoly (ethylene glycol) carboxylic and acid amikacin to hyperthermia, significantly reducing biofilm formation by P. aeruginosa.

45Another well-established approach for microbial control is the photodynamic therapy (PDT). In this case, a photosensitizer agent is used to produce ROS or singlet oxygen when activated by light irradiation (typically UV–vis or near infrared light) in the presence of oxygen, since these species eradicate microorganisms via oxidative damage of cell wall or membranes, cytoplasm leakage, lipid peroxidation, metabolic inhibition and also damages to mitochondria, membrane transport system or DNA. coated negatively charged AgNPs (−40 mV) with positively charged polyethylenimine (PEI) and chlorin e6 (Ce6), a natural molecule approved by FDA and commonly used as a photosensitizer. Although AgNPs and PEI-Ce6, in separate, were able to reduce the microbial adhesion, when combined, this system completely inhibited the biofilm of E. coli formation and significantly reduced S. aureus biofilm formation. The exposure of biofilms of S. aureus and E. coli to AgNPs-PEI-Ce6 suspensions in combination to PDT significantly reduced the microbial survival in vitro, and in mice both PEI-Ce6 and AgNPs-PEI-Ce6 promoted a faster recovery of S. aureus wound infection, showing the benefits of combining NPs and PDT.

46To mimic real-world infections more accurately, researchers, utilized biofilms derived from clinical isolates. These biofilms often harbor multiple bacterial strains, requiring antibiofilm agents with diverse modes of action. Similarly, to treating standard reference strains, studies have shown that AgNPs are effective against biofilms formed by clinical isolates, demonstrating similar success to those observed with standard lab strains. For instance, investigated the impact of caries on oral biofilms (primarily composed of S. mutans and S. sobrinus). They found that AgNPs were more effective in controlling biofilms from individuals without caries.

47The scenario of AgNPs in biofilm control shows that, despite the well-known properties of these NPs, the combination with other compounds or approaches can be a good option in the development of antibiofilm agents that efficiently control the formation or eliminate these bacteria structures, even when involving resistant strains.

48The fight against infections associated with the previously mentioned bacterial biofilms, particularly those caused by MDR strains has driven the development of novel approaches. Persistent bacteria exhibit phenotypic changes that enable them to evade antimicrobial agents, often through genetic mutations. Among the various bacterial species described in the literature, Gram-negative bacteria have shown the most significant resistance to silver. This resistance mechanism is partly due to presence of lipopolysaccharides (LPS) on their cell wall, which induce electrostatic repulsion with negatively charged AgNPs. Factors contributing to silver resistance in bacteria involve chemical detoxification mechanisms and active efflux systems. These systems, such as P-type ATPases and cation/proton transporters facilitate the transport of silver ions into or out of bacterial cells. Additionally, silver ions can be reduced to their inactive elemental metallic form, decreasing their antimicrobial activity.

49Like the mechanism of action of AgNPs as an antimicrobial agent involves a series of factors and multiple ways of interacting, studies suggest that the mechanism of bacterial resistance to silver involves a combination of interrelated systems. Among the main mechanisms of bacterial resistance to silver described in the literature, the following may occur: induction of aggregation of silver nanoparticles; reduction of Ag+ ions to Ag0; contact protection and prevention of silver entry into cells; efflux of silver nanoparticles and Ag+ ions through cellular efflux pumps; activation of defense mechanisms and repair of cellular damage; biofilm defense; antibiotic-mediated silver resistance; mutation; modulating the surface charge of cell membranes and response to stress and vitality.

50Exposure to sublethal concentrations of AgNPs can trigger the development of resistance phenotypes due to oxidative stress and DNA damage. These phenotypes may arise through active and passive resistance mechanisms. Active resistance mechanisms involve the direct response of the bacterium to the antimicrobial stimulus, including the use of efflux pumps and genetic mutations. Passive resistance mechanisms involve intrinsic characteristics of the bacterium, such as the thickening of the cell wall and the formation of bacterial biofilm.

51Factors contributing to silver resistance in bacteria involve chemical detoxification mechanisms and active efflux systems. These systems, such as P-type ATPases and cation/proton transporters, play a role in transporting silver ions into or out of bacterial cells. Additionally, it is possible to reduce the silver concentration by converting the Ag+ ion to the inactive elemental metallic form Ag0, thus not presenting antimicrobial activity and other properties associated with the silver ion.

52AgNPs can interact with nucleic acids, aiding in the aggregation, formation, structure, and integrity of bacterial biofilm, as well as with membrane proteins and enzymes that promote homeostasis, as well as polysaccharides and lipids. These interactions can originate hydrophobic, Van der Waals, hydrogen bonding, π–π, ionic, or electrostatic, with the latter contributing most to the biocidal activity of nanoparticles.

53The ability of bacterial resistance to AgNPs was evidenced through TEM and UV–visible spectroscopy of nanoparticles. In the case of the E. coli CCM 3954 strain, classified as ‘silver-resistant’, aggregation and/or precipitation of AgNPs were induced. Flagellin, a structural protein of bacterial flagella responsible for bacterial locomotion, played a role in promoting the aggregation of AgNPs, resulting in the reduction of their antibacterial effect.

54A fact with few studies yet relates that resistance to antibiotics in bacteria can also lead to the development of resistance to silver. A study demonstrated that both AgNPs and Ag+ can remove mature P. aeruginosa biofilms depending on the concentration of these compounds, however, AgNPs and Ag+ at the same concentrations were not able to destroy the biofilm formed by gentamicin-resistant P. aeruginosa. Therefore, the use of inorganic antimicrobial agents and antibiotics needs to follow more rigorous protocols, as improper use can lead to the emergence of superbugs and compromise the action of various antibiotic therapy strategies.

55Researchers studied the heredity and stability of resistance to AgNPs. By removing and reintroducing AgNPs in various growth passages, the hypermotile E. coli strain K-12 MG1655 maintained resistance, indicating a hereditary, irreversible, and permanent genetic alteration due to the expression of resistance genes, even in the absence of silver. Genomic analyses identified a missense mutation (R292L) in the cusS gene, which remained constant in all lineages. This mutation, located in the active site of dimerization and the histidine phosphotransfer domain, enhances silver ion efflux, thereby conferring resistance to AgNPs.

56Analyzing the impact of bacterial motility on AgNPs resistance, researchers found that non-motile E. coli strain did not exhibit significant MIC increase upon repeated AgNPs exposition. Genomic analysis did not reveal the presence of resistance genes, indicating that resistance to AgNPs may be specific to strains with bacterial motility.

57To assess physiological changes triggered by AgNPs exposure, E. coli pre-treated with AgNPs showed increased MIC and MBC values (of two to eight times) when subsequently exposed to antibiotics like aminoglycosides, penicillins, and phenicols compared to the group that did not receive pre-treatment with AgNPs, indicating adaptive responses mechanism that promove antimicrobial resistance.

58Most studies determining the effectiveness of AgNPs as antimicrobials are performed in single-species cultures. However, bacteria are rarely found in a single-species group in the environment. Researchers have demonstrated mechanisms that allow the sharing of resistance between bacteria that are usually found in association. For example, multiple compounds secreted by P. aeruginosa have been found to increase the tolerance of S. aureus to silver, both microorganisms are found associated in severe chronic infections leading to increased virulence and tolerance to antibiotics. The secretion of Pseudomonas quinolone signal (PQS) or compounds directly controlled by PQS, as well as the amino acids serine and threonine, generate silver tolerance in S. aureus. These compounds probably affect the physiology and metabolism of S. aureus (PQS, serine and threonine) by decreasing the availability of silver in the medium through bonds.

59Understanding these complex interactions between bacteria, AgNPs and silver ions is crucial for developing effective strategies against bacterial resistance. Such insights contribute to significant advancements in nanomedicine and infection control strategies.

60Proteomics is a branch of molecular biology that studies the set of proteins expressed by an organism or cell in response to different conditions. For instance, when microorganisms are exposed to a certain antimicrobial agent such as AgNPs, proteomic analysis provides information about action and resistance mechanisms of these microorganisms to the antimicrobial agent.

61Recent findings have revealed molecular processes underlying the action of AgNPs against microorganisms. Understanding these proteomic mechanisms can guide the development of new antimicrobial strategies. Here are some relevant considerations regarding the proteomics of microorganisms exposed to AgNPs which aid in understanding the results of such analyses.

62Proteomic analysis allows for the identification and quantification of specific proteins from a given organism or cell. Generally, each microorganism has a distinct identity (fingerprint) in its protein expression profile. When microorganisms are exposed to AgNPs, it is possible to investigate how these nanoparticles affect protein expression. Techniques such as two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry are frequently used to analyze the proteomic profile.

63The study of microbial proteomics opens a universe of possibilities; different bacterial strains can exhibit different protein expression profiles even when exposed to a single formulation of silver nanoparticles. Similarly, a standard strain may display distinct proteomic profiles when exposed to different AgNPs. AgNPs are generally considered a unique type of antibacterial agent, but their physical and chemical properties determine the way they interact with bacterial cells, the mode of action, and the response of the exposed bacterial cell.

64AgNPs can interact with cellular proteins, leading to changes in protein expression. They can affect metabolic pathways, signaling processes, and stress responses. Proteomic analysis contributes to the identification of specific proteins involved in these mechanisms. Table 4 represents the data of proteins expressed in bacteria after treatment with AgNPs.

65In early reports of proteomic analyses, researchers identified a potential mode of action underlying the antibacterial action of AgNPs. An accumulation of protein precursors from the envelope was found in the proteomic signatures of E. coli cells treated with AgNPs, suggesting that AgNPs may interact with the bacterial membrane. Brief exposure of E. coli cells to AgNPs resulted in changes in the expression of envelope proteins (OmpA, OmpC, OmpF, OppA, MetQ) and heat shock proteins (IbpA, IbpB, and ribosomal subunit 30S S6). Additionally, AgNPs and Ag+ ions in the form of AgNO3 exhibited similar mechanisms of action targeting the membrane. AgNPs and Ag+ ions were used at nanomolar and micromolar levels, respectively, suggesting that AgNPs have significantly greater antimicrobial activity than Ag+ ions.

66Studies conducted with P. aeruginosa (Gram-negative bacteria) treated with AgNPs yielded comprehensive proteomic responses, aiding in the elucidation of the antimicrobial mechanism. Proteomic analysis of bacteria treated with AgNPs revealed elevated levels of enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as alkyl hydroperoxide reductase and hydroperoxide resistance proteins. There was also significant positive regulation of low-oxygen regulatory oxidases, such as subunits P2, N2, and O2 of cytochrome c oxidase type cbb3. This demonstrates that the primary mechanism of action of AgNPs involves the imbalance of oxidation and antioxidation processes and the failure to eliminate excess ROS. Another study identified 5 silver-binding proteins and 59 proteins regulated by silver (27 positively regulated proteins and 32 negatively regulated proteins). Proteomic profiling showed that the cell membrane was the primary target of AgNPs, leading to oxidative stress induced by ROS generation. The release of silver ions and specific particle effects synergistically contribute to the antibacterial action of AgNPs. Moreover, the same silver-binding proteins were obtained with AgNPs and silver ions, indicating that AgNPs likely affect the cell membrane and react with proteins, releasing silver ions. These results demonstrate that the antimicrobial activity of AgNPs is due to the synergistic action of dissolved silver ions release and specific particle effects.

67In another investigation, proteomic analysis of AgNPs against multidrug-resistant P. aeruginosa analyzed antibiofilm mechanisms. Based on Tandem Mass Tag (TMT)-labeled quantitative proteomics, various antibiofilm mechanisms of AgNPs were revealed. These included inhibition of adhesion and motility, strong stimulation of oxidative stress response, disruption of iron homeostasis, blocking of energy production (both aerobic and anaerobic), and affecting quorum sensing detection. The latter is a communication mechanism used by bacteria to coordinate gene expression based on their population density. Proteomic analysis showed alterations in protein expression at different functional levels, suggesting that AgNPs may eliminate P. aeruginosa biofilms in multiple ways.

68Based on quantitative proteomic analysis (iTRAQ), it was discovered that AgNPs disrupt the natural morphology environment of S. suis and its biofilm. Several proteins related to peptidoglycan synthesis were negatively regulated, including penicillin-binding proteins (PBPs), glycosyltransferases, and LytR family transcriptional regulators, leading to bacterial membrane rupture. Additionally, AgNPs can prevent bacterial adhesion, interfere with QS system, and inhibit bacterial growth by targeting the cell division protein FtsZ and the chromosomal replication initiator protein DnaA. Increased oxidative stress is also a relevant factor contributing to bacterial death.

69Conventional antibiotics have revolutionized the treatment of bacterial infections and led to the emergence of strains resistant to various antibiotics restricting antimicrobial molecules to hospital to avoid the development of superbugs that cannot be combated with the existing arsenal of antibiotics. The continuous and excessive use of such antibiotics has led to outbreaks of superbugs in hospitals and communities. In recent decades, silver has been used in medical treatments such as burns, wounds, and bacterial infections. Metallic silver, silver nitrate, and silver sulfadiazine have been used in these treatments. Nowadays, AgNPs and silver ions are used as antibacterial agents in the medical field in the form of nanoparticles and ions, with proven effectiveness against bacteria, fungi, and viruses. These nanostructures can still be modified or functionalized to be more effective than pure AgNPs. Size, shape, concentration, surface charge, and functionalization are the main properties that determine antimicrobial efficiency. The broad-spectrum antimicrobial activity of nanomaterials compared to antibiotics may off a solution to combat MDR bacterial strains. For example, AgNPs act through various mechanisms, including overcoming antibiotic resistance mechanisms, damaging membranes, and preventing biofilm formation. When applied together with other antimicrobial molecules, therapy can be enhanced and the emergence of new MDR strains avoided.

70Spherical AgNPs showed efficacy against Porphyromonas gingivalis and S. mutans (MIC = 8 μg/mL) and significantly prevented the adhesion of S. mutans in vitro. Proteomic analysis showed that after a short period of treatment of bacteria with AgNPs, changes in the expression of various heat shock proteins and bacterial cell coating proteins were observed, suggesting that nanoparticles may penetrate the bacterial membrane leading to destruction. AgNPs can also cause a decrease in intracellular potassium, resulting in a reduction in ATP levels. Possible molecular targets of AgNPs may be thiol protein groups and the phospholipid part of the bacterial cell membrane. Therefore, AgNPs@Abelmoschus esculentus may be good candidates for oral hygiene agents to prevent periodontopathic conditions and dentures.

71Researchers studied a strategy combining hollow mesoporous silica nano spheres (HMSN) loaded with AgNPs, vancomycin and hemin (HAVH) for the elimination of MDR bacteria in abscess therapy. Demonstrating that the multi-path antibacterial mechanisms of HAVH could reduce the risk of drug resistance development by addressing the limitation of conventional antibiotics. In addition to the strategy combining AgNPs with antibiotics (HAVH), NIR irradiation showed improved synergistic effects, suggesting the potential of combinatory strategies for treating MDR bacterial infection. Enrichment analysis of genetic ontology (GO) indicated that differentially expressed proteins were associated with biological processes, cellular components, and molecular functions, including changes in nucleotide binding sites, cytoplasm, and transcription processes.

72Prolonged exposure to AgNPs can lead to the selection of resistant microorganisms. Proteomics can reveal how microorganisms adapt to AgNPs by modifying their protein expression. Thus, application studies should consider the possibility that AgNPs may increase antibiotic resistance by causing mutations in resistance genes. A study using E. faecalis demonstrated the ability of these microorganisms to develop resistance to AgNPs. This resistance consists of the expression of multifaceted mechanisms. Proteomic analysis revealed that 1,080 proteins were unique to E. faecalis exposed to AgNPs compared to 8 identified in the control sample. Among the affected biological processes and molecular functions are oxidative stress (superoxide dismutase and peroxiredoxin AhpC); transcriptional factors (Cro, Lux, and Rex); universal stress protein; and fibronectin adhesin Pav-type (FIBR). Therefore, strategies should be explored to prevent the emergence of this type of resistance, which, when combined with co-stimulation of antibiotic resistance, could increase the emergence of bacteria and infections resistant to antimicrobial drugs. To avoid resistance generation against AgNPs, the study suggested the combined use of nanoparticles prepared with different metals to prevent the spread of resistant strains.

73The transfer of antibiotic resistance genes (ARGs) is considered one of the main ways of spreading resistance, which occurs through horizontal gene transfer (HGT). There are reports that AgNPs enhance the transfer of ARGs via plasmid transmission between bacterial genera. It was determined that both AgNPs and ionic silver, in environmentally relevant and sublethal concentrations, facilitate the transfer of the IncP-α RP4 plasmid produced and supplied by Escherichia coli K-12 LE392 to the recipient bacterium Pseudomonas putida KT2440. Based on proteomic analyses, genetic sequencing, the potential ecological risks of environmental levels of AgNPs and silver ions in the promotion and dissemination of ARGs by HGT were exposed. Thus, warning against the excessive use of AgNPs in personal care formulations as antimicrobial agents.

74Among the metallic nanoparticles consumed daily, AgNPs have become the most common due to their antimicrobial effect. A study concerned with the effects of sublethal concentrations of AgNPs on intestinal biofilms investigated the in vitro proteomic response of a simulated intestinal biofilm with E. coli monospecies to chronic and acute exposures of sublethal concentrations of AgNPs. Of the 1,917 proteins identified, 212 showed different levels of expression. Several pathways were altered, including biofilm formation, bacterial adhesion, response to reactive oxygen species stress, and glucose utilization. These findings contribute to a better understanding of the molecular basis of the antibacterial activity of AgNPs, highlighting potential targets for the development of new antimicrobial agents or new therapies that combine classical antimicrobial agents and nanoparticles. Thereby, improving the antimicrobial arsenal against infections caused by multidrug-resistant microorganisms affecting humans and animals. However, alongside these advancements, it is crucial to consider the potential impact on the natural microbiota, as well as the interaction of AgNPs with other cells and biological structures of organisms. Since AgNPs are known to significantly impact living cells, their widespread use raises concerns about both immediate and long-term effects following exposure. For example, a proteomic study with macrophages showed that some functions of these specialized cells, such as lipopolysaccharide-induced cytokine production and nitric oxide, did not return to baseline even 72 h after exposure, showing that some effects of AgNPs persist even after exposure has ceased. This process of recovery of homeostasis after acute exposure to AgNPs involves hundreds of proteins and an enormous energy consumption, 50% higher than normal. The persistence of changes in the proteomic profile may be related to the intracellular silver persistence during the recovery period. Although the concentration of cellular lethality is not reached, post-exposure effects can impact the health status of living organisms. Proteomic analysis has become a valuable tool for identifying new biomarkers with greater sensitivity and specificity, contributing to a better understanding of disease progression, the development of new drugs and combined therapeutic strategies, as well as the improvement of toxicological studies. The identification and quantification of proteins in cells, tissues, or organisms provide insight into the activity of new therapeutics at specialized and personalized molecular levels.

75Silver nanoparticles (AgNPs) have emerged as a promising antimicrobial agent, exhibiting potent activity against a broad spectrum of microorganisms, facilitated by a multifaceted mechanistic approach that enables them to interact with microbial cells in multiple ways. Proteomics has played a crucial role in elucidating the mechanisms of action of AgNPs at the molecular level, revealing that they disrupt cellular processes, including protein synthesis, membrane transport, and cell signaling pathways. These insights have been pivotal in understanding the multifaceted antimicrobial mechanisms of AgNPs. The use of AgNPs as antimicrobial agents offers a promising solution to combat the growing threat of antibiotic resistance, a global health concern and their applications in medical devices, wound healing, and water treatment are being explored.

76Future perspectives for AgNPs as antimicrobial agents lie in their potential to be combined with other therapeutic agents, such as antibiotics and antioxidants, to enhance their efficacy and reduce toxicity. Emerging trends include the development of targeted AgNPs that can selectively target specific microorganisms and reduce off-target effects. Additionally, the application of AgNPs in understanding microbial proteomics and identifying novel antimicrobial targets presents a thrilling research avenue, particularly in the face of the escalating global crisis of AMR. As AMR poses a significant threat to the effectiveness of conventional antibiotics against bacterial infections, the WHO’s Global Antimicrobial Resistance and Use Surveillance System (GLASS) has reported alarmingly high resistance rates among common bacterial pathogens, underscoring the urgent need for innovative solutions. Therefore, the potential of AgNPs to revolutionize the field of antimicrobial therapy, and further research is required to fully explore their potential and address the challenges associated with their use.

77AR: Conceptualization, Writing – original draft, Writing – review & editing. JB: Conceptualization, Writing – original draft, Writing – review & editing. MR: Writing – original draft, Writing – review & editing. VT: Writing – review & editing, Writing – original draft. LM: Supervision, Funding acquisition, Writing – review & editing. PL: Supervision, Funding acquisition, Writing – review & editing. AL: Funding acquisition, Supervision, Conceptualization, Resources, Writing – review & editing.