Nanoparticles are used as antibacterial agents (Morones et al., 2005; Ruparelia et al., 2008; Varshney et al., 2012). The composition of antibacterial nanoparticles varies in physical and chemical properties. Some employ organic based liposomes and capsules filled with conventional antibiotics or novel RNAs, termed nano-carriers; while others exploit the cation leaching from metal colloid surfaces as the main antibacterial agent (Zhang et al., 2010; Hallaj-Nezhadi and Hassan, 2015; Mi et al., 2018). In the latter class of nanoparticles, the metal colloid can be engineered to include different chemical compositions (such as silver or copper), surface functionalities (such as aqueous suspension stabilizing agents or surface charges), or primary particle size (<15 nm in diameter to enable passive diffusion across the bacterial cell wall and other intracellular membranes or > 50 nm to enable extended duration of cation leaching in either biological or environmental matrices). Each of these properties are compiled into a nanoparticle profile and if the antibacterial activity of each property can be measured, then antibacterial nanoparticles can be designed in a safe and effective manner.

Silver and copper nanoparticles are commercially available and are currently used as antibacterial (and in some cases antimicrobial) applications. For instance, silver particles are incorporated into bandages to prevent bacterial infections in wounded skin (Wilkinson et al., 2011; Liu et al., 2018). Copper nanoparticles are used in antifouling paints for the prevention of biofilm formation in the hull of ships (Anyaogu et al., 2008; Padmavathi et al., 2019). In some cases, the nanoparticle formed is a zero-valent metal colloid which can be described as clusters of metal ions reduced to zero valency and self-assembled into spheres coated with a blanket of positively, negatively, or neutrally charged functional groups (Li et al., 2008; An and Somorjai, 2012). Metal-based “core-shell” nanoparticles are precisely engineered for size, shape, and surface charge – three of the most useful and tailorable properties that induce cytotoxicity (Berg et al., 2009; El Badawy et al., 2011; Mélinon et al., 2014; Liu et al., 2021).

In other cases, metal nanoparticles can be produced in a crystallization process involving both a metal cation and inorganic anion (Ahmed et al., 2012). One example is copper (I) iodide (CuI). CuI are suspected to have enhanced antimicrobial activity because both copper and iodine ions are present and have the potential to leach into the surface matrix (Renné et al., 2017). Copper ions, in particular the monovalent form (Cu⁺¹) is cytotoxic to bacteria, viruses, fungi, and molds due to oxidation process [i.e., from its monovalent to its divalent (Cu⁺²) and the associated generation of hydrogen peroxide in the presence of atmospheric oxygen or water (Tamayo et al., 2016)]. Interestingly, this is the same biological mode of action when using high-proof alcohol (arguably one of the most used method of sterilizing surfaces) (Palmer, 2010).

The need to address the issue of antimicrobial resistance due to broad use and abuse of antibiotics is very critical because of increased occurrence of multi-drug resistant bacterial strains which contribute to the reemergence of infectious diseases and persistent spread of hospital acquired infections. Antimicrobial resistance develops either through new mutation and selection in previously susceptible species, or by horizontal gene transfer during conjugation, transduction, and transformation in which new genetic materials are acquired from other resistant organisms (McManus, 1997). The new sets of antibiotics developed in the last decade have the same mechanism of antibacterial action as older antibiotics and have not been able to decrease the incidence of antibiotic resistance (Quinteros et al., 2016). This has necessitated the adoption of alternative strategies to treat bacterial infections rather than depending completely on conventional antibiotics which also have the limitation of low bioavailability and poor penetration at sites of infection (Canaparo et al., 2019). Metal nanoparticles can also be used as delivery agents when functionalized with a conventional antibiotic. In addition to combating antimicrobial resistance, this can also improve antimicrobial treatment efficiency, increase drug concentration at infection site, decrease bioaccumulation and decrease the risk of toxicity to nontarget tissues (Miller et al., 2014).

The application of nanotechnology in the fight against antimicrobial resistance could proffer solutions to some of the critical factors responsible for the persistence of antimicrobial resistance (Shaikh et al., 2015). Metals such as silver and copper have antibacterial properties in the bulk form and can be used as alternative forms of antibacterial therapy in nano-formulations which have unique physicochemical properties (Morones et al., 2005; Ruparelia et al., 2008; Varshney et al., 2012). The large surface area to volume ratio of nanoparticles creates active sites for attachment with different functional groups in a biological environment (Wang et al., 2006; Ezeuko et al., 2021). Nanoparticles serve as excellent candidates for drug delivery when paired with active pharmacological ingredients (Patra et al., 2018; Ezeuko et al., 2021). If nanoparticles are a viable alternative to conventional antibiotics, then progress must be made to ensure responsible development of engineered nanoparticles at environmentally friendly concentrations. For example, it is postulated that the ideal use of nanoparticles in agricultural, medical, and environmental applications is administration in lower doses because at higher doses, unintended consequences may become a significant factor (Roguska et al., 2015; Khan et al., 2016; Graham et al., 2017). In fact, there are discrepancies in literature when assessing the effectiveness of metal nanoparticle toxicity to bacteria (and other microorganisms). Most published reports focus on very high concentrations in an effort to prove efficacy (Roguska et al., 2015). However, we hypothesize that if nanoparticles are designed to specifically increase cytotoxicity in bacteria, while other environmental and health effects are reduced, then the doses needed to demonstrate metal-based nanoparticle antibacterial activity can be lowered.

This study reports a systematic investigation designed to deconvolute the relative bacterial inhibition of the physicochemical properties of four different engineered metal nanoparticles. We used minimum inhibitory concentration (MIC) coupled with minimum bactericidal concentration (MBC) and viable count assays to study the nanoparticle sphere (silver versus copper), surface coating (a.k.a. stabilizing agent; CTAB versus PVP), surface charge (positive versus neutral), and dose–response.

The sample naming convention used in this study is as follows: silver nanoparticles coated with CTAB are termed ‘CTAB-AgNPs’, silver nanoparticles coated with PVP are termed ‘PVP-AgNPs’, copper nanoparticles coated with CTAB are termed ‘CTAB-CuNPs’, and copper nanoparticles coated with PVP are termed ‘PVP-CuNPs’. In addition, the following controls were used: ‘0.4% CTAB’ representing the estimated concentration of CTAB in the CTAB-stabilized nanoparticles and ‘0.4% PVP’ representing the estimated concentration of PVP in the PVP-stabilized nanoparticles. These controls were used to parse the impact of the stabilizing agents independent of the nanoparticles. Lastly, the experimental design included a ‘no treatment’ control.

Synthesis of metal nanoparticles via chemical reduction is the most common method due to low cost and tailorable properties (Pham et al., 2012). During synthesis, nanoparticle physicochemical properties are controlled by varying the experimental parameters such as concentration of reactants, temperature, and reaction time. Synthesis of metal nanoparticles via chemical reduction method using the polymer polyvinyl pyrrolidone (PVP which confers a neutral charge onto the surface of the nanoparticles) and the surfactant cetyltrimethylammonium bromide (CTAB which confers a positive charge onto the surface of the nanoparticles) have been documented (Trigari et al., 2011; Pham et al., 2012; Verma et al., 2015). Surfactants and polymers have been employed as surface coating and surface stabilizing agents in the synthesis of metal nanoparticles because they protect the core of the nanoparticle from oxidation, prevent nanoparticle agglomeration due to electrostatic and steric repulsion, and are also used to control the particle size and shape (Sarkar et al., 2008; Pham et al., 2012; Roh et al., 2013). Nanoparticle characterization validated the presence of metal nanoparticles after the chemical reduction synthesis method. Transmission electron micrographs showed the shape and size of the particles while dynamic light scattering reported hydrodynamic diameter (HDD), zeta potential (ZP), and dispersity index (DI) (Zhang et al., 2020).

Stabilizing agents can inhibit bacteria growth. Cetyltrimethylammonium bromide (CTAB) inhibits bacteria growth. CTAB belongs to the class of quaternary ammonium compounds, and they are extensively used as antiseptics and disinfectants in residential and occupational settings due to their inherent bactericidal activities (Denyer, 1995; Liao et al., 2020; Ongwae et al., 2020). These quaternary ammonium compounds target bacteria cells via electrostatic interactions between the positively charged headgroup of the molecule and the negatively charged cytoplasmic membrane of bacteria cell. The electrostatic interaction initiates adsorption onto the bacteria cell followed by side chain permeation into the intermembrane region which causes cytoplasmic leakage and ultimate cell lysis (Jennings et al., 2015). Several studies have demonstrated that the surfactant CTAB has biocidal properties (Tamuly et al., 2013). Several outcomes have been shown to occur when bacteria cells are exposed to CTAB and other surface active agents, such as membrane adsorption, membrane disorganization, or release of cytoplasmic constituents into surrounding matrix (Salton, 1951; Salton et al., 1951). Silver and copper nanoparticles with CTAB induced more bacteria growth inhibition than CTAB alone. CTAB-AgNPs and CTAB-CuNPs decreased bacteria growth demonstrating that when CTAB was paired with the nanoparticles (either silver or copper), growth of bacteria colonies was substantially decreased or even eliminated in the case of S. multivorum.

The PVP-AgNPs and PVP-CuNPs were less effective in decreasing bacteria growth compared to the CTAB stabilized metal nanoparticles. In general, PVP-AgNPs were more effective than PVP-CuNPs and there were more viable colonies of S. multivorum exposed to PVP-CuNPs than the no treatment control. In contrast to the observed results here, other studies have shown high antibacterial efficacy of PVP-AgNPs but the antibacterial efficacy was attributed solely to the metallic silver core (Batista et al., 2018). In this current study, it was observed that the PVP stabilizing agent seemed to promote the growth of E. coli and S. multivorum; showing more viable bacteria colonies than the no treatment control group against these bacteria. This observation could be explained with the assumption that PVP acts as a growth promoting factor for bacteria. It has been shown that the high water-binding capacity of PVP aids the metabolism of cells by maintaining moisture content in the cell media and that PVP binds to bacteria toxins released into media during the stationary growth phase, thereby extending the time before they reach the death phase (Deaker et al., 2004; Biradar and Santhosh, 2018).

There were three independent assays used to ascertain the bacterial growth inhibition data. Results of all three assays confirm the findings. Table 2 summarizes the agreement among the assay results and provides a clear indication that CTAB stabilized metal nanoparticles are quite effective antibacterial agents and that S. multivorum was the most susceptible to nanoparticle antibacterial activity.

This study demonstrates the use of engineered metal nanoparticles as possible antibacterial agents. The recorded MIC and MBC values and viable counts of bacteria after exposure to surface stabilized particles show that they can be effective at low doses. Silver and copper nanoparticles have reactive surfaces when suspended in aqueous media and have been shown to produce reactive oxygen species (ROS) (Abdal Dayem et al., 2017; Ameh et al., 2022). These particles have demonstrated antibacterial action against a range of antibiotic resistant bacteria species such as clinical strains of methicillin resistant S. aureus and E. coli (Quinteros et al., 2016; Liu et al., 2020). Bacterial toxicity of metal nanoparticles has been reported to be dependent on a combination of factors such as dose, physical and chemical properties, and amount of bacteria (Hajipour et al., 2012). Nanoparticles that do not aggregate or agglomerate have more available surface area; more available surface area translates into more ROS generation, greater chance of bacteria cell membrane interaction, and increased levels of released metal ions in surrounding matrix (Pramanik et al., 2012).

The need to explore non-traditional methods of antibacterial therapy is critical due to the persistence in antimicrobial resistance and the utility of antimicrobial nanoparticles could be a beneficial tool in addressing this clinical problem of critical importance. The safety and efficacy of these nanoparticles should continue to be investigated via cell based in vitro pharmacokinetic studies to broaden the knowledge perspectives on their use in biomedical applications. Unknown and unintended consequences of the increased use of nanoparticles in the industrial, consumer and healthcare sectors pose key challenges to human and environmental health. Developing frameworks for assessing risk associated with nanoparticle exposure are integral tools for maintaining human health and environmental safety. The critical quality attributes of nanoparticles during their life cycle such as fate and transport parameters, human and environmental health hazard profile, best practices for safe production as well as use and disposal, must be considered when applying control measures to reduce the associated risk from nanoparticle exposure (Warheit et al., 2008). Thus, it is of utmost importance to develop quantitative methods of risk assessment for determining environmental levels of metal nanoparticles and the resultant health effects of nanoparticle exposure in order to manage and mitigate such risks.

The antibacterial efficacy of surface-stabilized metal nanoparticles was assessed to determine minimum inhibitory concentration and minimum bactericidal concentration. The antibacterial effects revealed in this study are dependent upon type of stabilizing agent and the interaction between stabilizing agent and the metal composition of the nanoparticle. The surface stabilized nanoparticles induced antibacterial action at relatively low doses. We have now assessed the potential for nanoparticles to be used as antibacterial agents in two different ways: antibacterial effects as a function of nanoparticle surface charge and antibacterial effects as a function of nanoparticle stabilizing agent. These features cannot be teased apart, but the knowledge unveiled herein can aid in formulating decisions. There is a need to further explore this phenomenon by systematically testing stabilizing agents that all confer a positive surface charge (like that of CTAB) or a neutral surface charge (like that of PVP) to differentiate effects between stabilizing agent and surface charge.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

TA: conceptualization, methodology, investigation, writing–original draft, formal analysis, and visualization. KZ and JD: methodology, investigation, and validation. WB: conceptualization, formal analysis, writing–review and editing, and visualization. CS: conceptualization, formal analysis, resources, writing–original draft, writing–review and editing, visualization, supervision, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

This research was supported by a cooperative agreement (#AP19PPQS&T00C023) between USDA Animal and Plant Health Inspection Service and Baylor University.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.