Escaping the ESKAPE Antimicrobial Resistant Cycle with EVQ-218

TL;DR

EVQ-218, a silver-based antimicrobial, shows resistance-resilient activity against dangerous drug-resistant bacteria, offering a potential solution to the growing problem of antimicrobial resistance.

Contribution

EVQ-218 demonstrates sustained antimicrobial activity without resistance development, unlike conventional antibiotics.

Findings

EVQ-218 did not show increased MIC or resistance indicators during serial passage experiments.

Bacteria resistant to tobramycin or ciprofloxacin remained susceptible to EVQ-218, indicating no cross-resistance.

EVQ-218 acts through a non-lytic, intracellular mechanism targeting sulfur-associated biomolecules.

Abstract

Background/Objectives: Antimicrobial resistance (AMR) continues to expand under sustained exposure to conventional antibiotics, contributing to the emergence of multidrug- and pan-resistant bacterial pathogens. There remains a critical need for antimicrobial agents that maintain activity during prolonged selective pressure while minimizing the potential for resistance development. This study aimed to evaluate EVQ-218, a non-ionic silver-based antimicrobial, against World Health Organization-designated ESKAPE pathogens. Methods: EVQ-218 was assessed using extended serial passage experiments performed under both sub- and supra-minimum inhibitory concentration (MIC) conditions. Comparative resistance selection experiments were conducted in parallel using tobramycin and ciprofloxacin, and susceptibility was evaluated through MIC determination and phenotypic analysis. Results: Across extended serial passage experiments, EVQ-218 did not exhibit measurable increases in MIC or phenotypic indicators of adaptive resistance. In contrast, parallel exposure to tobramycin and ciprofloxacin resulted in rapid and sustained MIC elevation. Notably, isolates that acquired resistance to either comparator antibiotic retained susceptibility to EVQ-218, indicating a lack of cross-resistance. Mechanistic analyses were consistent with a non-lytic, intracellular mode of antibacterial activity involving disruption of sulfur-associated biomolecular processes, suggestive of a multi-site mechanism distinct from classical antibiotics. Conclusions: These findings support EVQ-218 as a promising broad-spectrum antimicrobial candidate with resistance-resilient activity and warrant further investigation of its potential role in addressing unmet needs in AMR therapeutics.

Figures

Full text

1. Introduction

Antimicrobial resistance (AMR) has emerged as one of the most formidable threats to global health, driven by decades of excessive and inappropriate antibiotic use across clinical, agricultural, and environmental settings [1,2]. In 2024, the World Health Organization (WHO) underscored six of the most critical antibiotic-resistant pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., collectively known as the ESKAPE pathogens, for their ability to “escape” the effects of nearly all available antimicrobial agents. These pathogens account for a substantial proportion of nosocomial infections and are responsible for increasing morbidity, mortality, and healthcare costs worldwide [1,3,4].

The ESKAPE pathogens exemplify the complexity of AMR, representing both Gram-positive and Gram-negative bacteria that deploy multiple resistance strategies, including (i) target site modification preventing drug binding, (ii) efflux pump activation expelling antibiotics, (iii) enzymatic degradation or modification of drugs, (iv) reduced outer membrane permeability, and (v) biofilm formation providing a physical and metabolic barrier to treatment [5,6,7]. These adaptive mechanisms are often compounded by genetic recombination, horizontal gene transfer, and regulatory network alterations that promote resistance evolution and persistence under selective pressure [8].

Confronting AMR demands a paradigm shift toward therapeutics that can bypass or dismantle these bacterial defense systems. Such innovations must overcome key resistance determinants, including cell envelope impermeability, enzymatic inactivation, and biofilm resilience, while limiting the emergence of new resistance phenotypes. Nanomedicine and nanoparticle-based therapeutics have recently emerged as promising strategies, offering multimodal mechanisms of action capable of circumventing traditional resistance pathways [9,10,11,12,13]. Within this context, the development of novel antimicrobials that maintain efficacy without promoting resistance evolution represents an urgent scientific and clinical priority [8,14,15].

To highlight the critical need today, ventilator-associated pneumonia (VAP) represents one of the most severe and costly manifestations of anti-microbial resistance in both hospital and community settings, contributing substantially to morbidity, mortality, and prolonged intensive care unit stays worldwide. In the United States alone, VAP remains among the most common hospital-acquired infections, with incidence rates ranging from 5–15 cases per 1000 ventilator days and attributable mortality estimates of up to 30–50% in high-risk populations. Globally, the burden of VAP continues to rise in parallel with increasing antimicrobial resistance and expanding use of invasive mechanical ventilation [16].

Notably, the pathogens most frequently implicated in VAP overlap extensively with the WHO-designated ESKAPE group, including Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, and Enterobacter spp. These organisms possess intrinsic and acquired resistance mechanisms that enable persistence on ventilator surfaces, within biofilms, and in the lower respiratory tract, rendering standard antibiotic regimens increasingly ineffective. As a result, VAP caused by multidrug-resistant ESKAPE pathogens remains exceptionally difficult to treat and is associated with poor clinical outcomes.

Current therapeutic strategies increasingly emphasize narrow or monospectrum antimicrobial agents targeting individual pathogens; however, such approaches may inadvertently permit the overgrowth of non-target organisms within polymicrobial pulmonary environments. In the context of respiratory infections, this selective pressure can exacerbate dysbiosis and facilitate secondary or breakthrough infections, particularly in critically ill or immunocompromised patients. Consequently, there remains a critical need for antimicrobial technologies capable of broadly suppressing pathogenic bacteria without driving compensatory dominance of alternative resistant species.

Despite advances in antimicrobial development, there is currently no universally accepted standard of care for preventing or mitigating pulmonary exacerbations associated with bacterial lung infections [17]. This is particularly frightening for patients with cystic fibrosis. Technologies that can function either as direct therapeutics or as preventative interventions (such as coatings, inhaled agents, or adjunctive antimicrobial strategies) represent a significant and largely unmet clinical need. Within this landscape, the development of resistance-resilient antimicrobial platforms with activity against the full spectrum of ESKAPE pathogens may offer substantial benefit for both hospital-acquired and non-hospital-acquired pulmonary infections.

1.1. Silver’s History

Despite the longstanding recognition of silver’s antimicrobial properties, relatively few silver-based active pharmaceutical ingredients have progressed into systemic or inhaled therapeutic development. This limited clinical translation largely reflects well-documented safety concerns associated with ionic silver (Ag^+^), including cytotoxicity, off-target protein interactions, tissue accumulation, and the risk of argyria following prolonged exposure. As a result, most clinical and late-stage development efforts involving silver have been restricted to topical formulations, wound dressings, or surface coatings, rather than systemically administered or pulmonary therapeutics.

Consequently, there are currently few, if any, silver-based antimicrobial agents in the clinical pipeline designed for direct treatment of invasive or pulmonary infections. This absence underscores a significant gap between silver’s potent antimicrobial activity and its safe therapeutic deployment. The development of silver-based technologies that decouple antimicrobial efficacy from ionic silver release therefore represents a critical unmet need and a key opportunity for innovation within the antimicrobial landscape.

1.2. Properties of EVQ-218

Nanoparticles are conventionally defined by the International Organization for Standardization (ISO) and ASTM as nanomaterial with all three external dimensions in the range of approximately 1–100 nm, a classification grounded primarily in dimensional criteria rather than specific biological or functional activity. At this scale, high surface area to volume ratios enhance chemical reactivity and biological interactions. Organic and carbon-based nanoparticles are predominantly used as drug delivery platforms, whereas inorganic metal and mineral nanoparticles, particularly silver nanoparticles (AgNPs), exhibit intrinsic antimicrobial activity, including biofilm disruption and bactericidal effects.

The antimicrobial efficacy of conventional AgNPs is inseparable from the release of silver ions (Ag^+^). Ag^+^ mediates membrane disruption, oxidative stress, enzyme inhibition, protein misfolding, and nucleic acid damage through electrostatic membrane interactions, cell wall penetration, and the generation of reactive oxygen species (ROS). Although effective, these mechanisms are associated with cytotoxicity, environmental persistence, and selective pressures that can promote stress responses, persistence phenotypes, and horizontal gene transfer. As a result, despite extensive study, conventional AgNPs remain constrained by safety and resistance-related limitations.

EVQ-218 represents a fundamentally distinct class of silver-based antimicrobial that departs from this ion-centric paradigm. EVQ-218 is a non-ionic silver nanoparticle composed of a metamaterial allotrope with physicochemical properties distinct from both ionic silver and surface-modified AgNPs [14,18]. Synthesized under high-temperature, high-pressure laser-based conditions, EVQ-218 forms ultra-uniform particles of approximately 5 nm (Figure 1) that adopt a smooth, spherical morphology characterized by shortened surface bonds and a stabilized surface topology. Scanning transmission electron microscopy coupled with electron energy loss spectroscopy (STEM–EELS) demonstrates a complete absence of surface oxygen, in contrast to NIST reference AgNPs produced by metal salt reduction, which exhibit substantial surface oxidation. This distinction has been previously reported and is independently reproduced here (Figure 2) [14].

The non-ionic nature of EVQ-218 has direct biological consequences. By preventing Ag^+^ release, EVQ-218 decouples antimicrobial activity from the cytotoxic mechanisms that define conventional silver nanomaterials. Consistent with this design, prior toxicological studies in RAW 264.7, MH-S, and THP-1 macrophage models revealed no detectable cytotoxicity at concentrations below established IC_50_ thresholds [18]. In vivo cytotoxicity studies, however, are yet to be validated.

EVQ-218 also exhibits exceptional long-term physicochemical stability across diverse solvents and under ambient light, maintaining an ultra-narrow size distribution without the need for surface coatings, stabilizers, or post-synthetic purification [14,18].

Importantly, EVQ-218 displays a biological mode of action that is mechanistically distinct from both antibiotics and traditional AgNPs. Rather than inducing membrane rupture, ROS generation, or catastrophic intracellular damage, EVQ-218 selectively disrupts protein disulfide bonds primarily within the bacterial periplasm. This process compromises essential structural and metabolic proteins while preserving cell envelope integrity (Figure 3). This non-lytic mode of action is hypothesized to minimize activation of stress-induced pathways associated with dormancy, persistence, and horizontal resistance transfer, thereby reducing the likelihood of resistance emergence.

EVQ-218 has demonstrated broad-spectrum efficacy against 64 clinically relevant isolates, including Gram-positive and Gram-negative bacteria, yeasts, filamentous fungi, and nontuberculous mycobacteria, in previous evaluations conducted at the Seattle Children’s Research Institute (SCRI) (Table 1) [17]. This study shows that EVQ-218 (Attostat Ag) exhibits consistent antimicrobial activity across Gram-negative, fungal, and NTM isolates, with MICs ≤ 2.0 µg mL^−1^ for most organisms and efficacy against all tested isolates at concentrations ≤ 10 µg mL^−1^, though BIC determination for NTM was not feasible with current methods. DF-STEM/EDS analysis reveals that EVQ-218 (Attostat Ag) enters cells, scavenges sulfur and is proposed to disrupt Fe–S clusters and disulfide bonds, thereby halting energy production and replication, with sulfur-to-EVQ-218 ratios distinct from analogous agents [19].

Building on these findings, the present study examines resistance development in ESKAPE pathogens following extensive and stringent serial passages under sub and supra minimum inhibitory concentrations (MIC) of EVQ-218. Parallel time-kill assays were conducted to determine whether EVQ-218 exerts bactericidal or bacteriostatic effects against these high-priority clinical isolates. STEM imaging further confirmed the absence of membrane lysis and revealed the accumulation of EVQ-218 within the periplasmic space, supporting a targeted mechanism of action. To assess cross-resistance potential, isolates resistant to tobramycin and ciprofloxacin, two frontline agents frequently used against multidrug-resistant respiratory infections, were tested against EVQ-218 [3,20,21]. Comparative analysis underscores EVQ-218’s preserved activity across resistant strains, highlighting its potential as a next-generation antimicrobial with durable efficacy.

2. Results

2.1. EVQ-218 Displays Broad-Spectrum Antibacterial Performance and Resistance Stability

To evaluate the potential for adaptive resistance, ESKAPE pathogens were serially propagated in the presence of EVQ-218 under increasing (Enhanced Resistance Study, P_0_ to P_20_) and stable (Baseline Resistance Study, P_0_ to P_30_) concentration regimes (Table 2). In the baseline resistance study, a single sub-MIC was used whereas in the enhanced resistance study, the MIC was doubled every 5 days (Table 2). In the enhanced resistance study, growth kinetics of four Gram-negative species, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii, were monitored over 24 h at passage zero (P_0_) and passage twenty (P_20_) across a concentration gradient ranging from 0.195 to 12.5 µg mL^−1^ (Figure 4).

In the enhanced study across all tested species, EVQ-218 produced a concentration-dependent suppression of growth, characterized by delayed onset of the exponential phase and reduced maximal optical densities relative to untreated controls. Complete inhibition of bacterial growth was observed at the respective MICs (3.13–12.5 µg mL^−1^) within the 24 h assay window (Figure 4). Sub-MIC exposures resulted in moderate growth delays but did not support recovery or adaptation over time.

Enhanced study comparison of passage zero (P_0_) and passage twenty (P_20_) isolates revealed no upward shift in MICs, indicating that repeated sub-MIC exposure to EVQ-218 did not promote tolerance or resistance development. P. aeruginosa maintained an MIC of 3.13 µg mL^−1^, while K. pneumoniae and A. baumannii remained stable at 6.25 µg mL^−1^ and 12.5 µg mL^−1^, respectively (Figure 4). E. cloacae also exhibited consistent inhibition at 12.5 µg mL^−1^, with no evidence of reduced susceptibility following serial propagation. The preservation of MIC values across twenty passages, together with consistent growth suppression kinetics, underscores EVQ-218’s resistance-resilient antibacterial mechanism.

Notably, P. aeruginosa, a pathogen renowned for intrinsic efflux systems and outer-membrane impermeability, displayed the lowest MIC among all tested strains, suggesting efficient nanoparticle penetration and interaction with periplasmic targets. In the baseline study, exposure of ESKAPE isolates to a stable sub-MIC (0.156 µg mL^−1^) over thirty passages (P_30_) similarly yielded no detectable shift in MIC (Supplementary Figure S1). Interestingly, MIC values were consistently lower when assays were performed in diluted cation-adjusted Mueller–Hinton broth (CAMHB), suggesting partial sequestration of EVQ-218 by proteinaceous or other analytes present in the medium. This interpretation was supported by internally replicated MIC experiments across graded broth concentrations, which demonstrated a concentration-dependent shift in apparent MIC values, consistent with medium-associated binding effects. Collectively, the uniformity of EVQ-218’s activity across genetically diverse Gram-negative pathogens, coupled with its stability under repeated sub-MIC challenge, highlights its potential as a broad-spectrum antibacterial agent with low risk of adaptive resistance or mutational escape.

The susceptibility of the Gram-positive ESKAPE pathogens Staphylococcus aureus and Enterococcus faecium to EVQ-218 was assessed across multiple nutrient conditions, including cation-adjusted Mueller-Hinton broth (CAMHB), Luria–Bertani (LB) broth, and brain heart infusion (BHI) broth. E. faecium exhibited limited proliferation in CAMHB, suggesting a dependence on nutrient-rich environments for optimal growth. Although the minimum inhibitory concentration (MIC) of E. faecium remained stable throughout serial propagation, the analysis was constrained by variable growth performance across media. In contrast, S. aureus displayed enhanced growth in LB relative to CAMHB, enabling more reliable assessment of antimicrobial susceptibility. Remarkably, in the baseline study, after thirty serial passages under sub-MIC exposure, S. aureus exhibited a two-fold reduction in MIC, declining from 5 µg mL^−1^ at P_0_ to 2.5 µg mL^−1^ at P_30_ (Supplementary Figure S2). This decrease may reflect either propagation-associated physiological decline or an adaptive increase in susceptibility to EVQ-218.

To evaluate the long-term stability of antibacterial efficacy in the enhanced study, growth kinetics of S. aureus and E. faecium were measured across a concentration gradient of EVQ-218 (0.195–12.5 µg mL^−1^) at P_0_ and P_20_ (Figure 5). Across all concentrations, EVQ-218 induced a clear, concentration-dependent suppression of growth, characterized by delayed onset of exponential phase and reduced maximum optical densities compared to untreated controls. At P_0_, S. aureus exhibited complete growth inhibition at 6.25 µg mL^−1^, with partial suppression at sub-MIC levels (1.56–3.13 µg mL^−1^) (Figure 5). E. faecium demonstrated greater tolerance, achieving complete inhibition only at 12.5 µg mL^−1^ (Figure 5).

Following twenty serial passages, no upward shifts in MICs were detected, indicating that prolonged sub-MIC exposure did not promote tolerance or resistance. S. aureus maintained stable inhibition kinetics but showed a marginal reduction in optical density at P_20_ (Figure 5), suggesting potential metabolic attenuation or heightened susceptibility. Conversely, E. faecium retained comparable growth dynamics between P_0_ and P_20_ (Figure 5), consistent with stable sensitivity to EVQ-218. Supporting our MIC results, we did not witness gross morphological effects (phenotypic) associated with changes in growth rate, cell envelope, surface modification, or adaptive resistance.

Together, these findings demonstrate that EVQ-218 maintains potent, durable antibacterial activity against clinically relevant Gram-positive pathogens, without evidence of adaptive resistance even after extended sub-inhibitory exposure. The observed stability and potential sensitization of S. aureus underscore EVQ-218’s promise as a resistance-resilient therapeutic candidate for multidrug-resistant Gram-positive infections.

2.2. EVQ-218 Exhibits Rate-Dependent Bactericidal Activity Against ESKAPE Pathogens

To characterize the relationship between EVQ-218 concentration and bacterial growth and death, time-kill assays were performed with six clinically relevant isolates. Bacterial cultures were exposed to EVQ-218 at concentrations above and below their respective MICs, and viable cells were enumerated every 30 min over a 3 h period (Figure 6). At 5 µg mL^−1^, all isolates except S. aureus were eradicated within 30 min, highlighting the rapid bactericidal effect of EVQ-218 at higher concentrations.

The Gram-positive strains were the most resilient, with measurable growth persisting until 150 min at the lowest EVQ-218 concentration (0.625 µg mL^−1^). Across all isolates, antimicrobial activity increased with concentration, consistent with a rate-dependent killing mechanism. The minimum bactericidal concentration (MBC) was less than four times the MIC for all strains, supporting classification of EVQ-218 as bactericidal according to established criteria. These observations suggest that EVQ-218 can effectively reduce bacterial burden in a concentration- and time-dependent manner.

Time-kill kinetics differed among strains, reflecting intrinsic susceptibility variations. Rapid initial reductions in colony-forming unit (CFU) were observed for P. aeruginosa, K. pneumoniae, A. baumannii, and E. cloacae, whereas S. aureus and E. faecium exhibited more gradual declines. At lower concentrations, bacterial counts either stabilized or decreased modestly, whereas higher concentrations produced pronounced killing, underscoring the importance of achieving sufficient drug exposure for maximal bactericidal effect.

Collectively, these results demonstrate that EVQ-218 exerts potent, rate-dependent bactericidal activity against a broad spectrum of ESKAPE pathogens. The concentration- and time-dependent nature of killing highlights its potential utility as a therapeutic agent, particularly against multidrug-resistant strains where rapid eradication is critical to clinical outcomes.

2.3. EVQ-218 Retains Activity Against Aminoglycoside and Fluoroquinolone-Resistant Isolates

To assess potential cross-resistance, EVQ-218 was tested against isolates resistant to tobramycin or ciprofloxacin. Within ten serial passages, resistance emerged in both Gram-negative and Gram-positive strains for the comparator antibiotics (Figure 7, Figure 8, Figure 9, Figure 10, Figure 11 and Figure 12). By contrast, P. aeruginosa resistant to either tobramycin or ciprofloxacin remained fully susceptible to EVQ-218, with MICs unchanged at 0.69 µg mL^−1^. Similarly, S. aureus displayed no cross-resistance, although growth in CAMHB remained limited, consistent with prior observations.

Notably, K. pneumoniae resistant to ciprofloxacin or tobramycin exhibited increased sensitivity to EVQ-218, with MICs of 0.34 µg mL^−1^ and 0.69 µg mL^−1^, respectively (Figure 9 and Figure 10). These findings suggest that acquisition of resistance through ribosomal or DNA gyrase/topoisomerase modifications rendered the bacteria more vulnerable to the multi-target activity of EVQ-218. MIC determinations were performed using eight concentrations spanning 0.078–10 µg mL^−1^, allowing quantification of changes over serial passages.

Time-resolved growth curves for representative isolates normalized to negative controls revealed that EVQ-218 consistently inhibited growth of resistant strains without evidence of cross-resistance (Figure 7, Figure 8, Figure 9, Figure 10, Figure 11 and Figure 12). The lack of cross-resistance underscores the distinct mechanism of action of EVQ-218 compared with conventional aminoglycosides and fluoroquinolones and highlights its potential to target multidrug-resistant pathogens.

2.4. Mechanism of Action of EVQ-218

Conventional silver nanoparticles (AgNPs) exert antibacterial activity through multiple mechanisms, including disruption of the cell wall and membrane, generation of reactive oxygen species, interference with intracellular biomolecules, and DNA damage. Ag^+^ ions released from AgNPs penetrate bacterial cell walls, causing leakage of cellular contents, and disrupt the peptidoglycan by altering β-1→4 glycosidic bonds and peptide cross-links. Electrostatic attraction between positively charged AgNPs and negatively charged membranes further promotes membrane adhesion, rigidity, and damage to lipids, proteins, and carbohydrates.

By contrast, EVQ-218 does not disrupt the native bacterial membrane (Figure 13, Figure 14, Figure 15, Figure 16 and Figure 17). Darkfield scanning transmission electron microscopy (STEM) revealed uniform intracellular distribution of EVQ-218 in both Gram-negative and Gram-positive bacteria, with notable accumulation in the periplasmic space (Figure 8). In P. aeruginosa, EVQ-218 localizes to the inner cytoplasmic membrane, whereas in K. pneumoniae, it is concentrated near the membrane (Figure 14). In Gram-positive S. aureus and E. faecium, energy-dispersive X-ray spectroscopy (EDS) demonstrated colocalization of silver and sulfur, indicative of a novel “silver–sulfur siege” (S^3^) mechanism (Figure 11 and Figure 12). Future research is warranted to gain a better understanding of the behavior exhibited by EVQ-218 and its prolonged antimicrobial effect.

Figure 13, Figure 14, Figure 15, Figure 16 and Figure 17 show intracellular localization of EVQ-218 and ultrastructural evidence for the silver–sulfur siege. High-resolution electron microscopy and elemental mapping reveal consistent intracellular accumulation of EVQ-218 across representative ESKAPE pathogens (Figure 15 and Figure 16), with localization patterns distinct from membrane-disruptive or lytic silver formulations.

2.5. The Silver-Sulfur Siege

Although EVQ-218 is non-ionic, its high-energy surface enables strong coordinative interactions with sulfur-containing biomolecules without releasing Ag^+^ ions. The S^3^ mechanism exploits the strong thiophilicity of EVQ-218, targeting sulfhydryl (–SH) groups, particularly in cysteine residues of bacterial proteins. This may lead to widespread disruption of proteins (e.g., Fe–S cluster-containing enzymes, oxidases, fumarases and ferredoxins) structure and function. Such multifaceted intracellular damage is consistent with global protein dysfunction and cellular collapse, likely contributing to broad-spectrum bactericidal activity of EVQ-218 [22,23,24,25].

To explore the physicochemical interactions underlying the “silver–sulfur siege,” the optical response of EVQ-218 was evaluated under varying chemical environments, including sulfur compounds, inorganic salts, and surfactants (Figure 18). In aqueous buffer, EVQ-218 displayed a sharp, intense absorption maximum at ~390 nm, characteristic of a well-dispersed, electronically coherent nanoparticle population. This resonance served as a baseline for assessing the stability and interaction profile of EVQ-218 under physiologically relevant and chemically perturbative conditions (Figure 18).

Exposure to sulfur-containing environments induced pronounced spectral perturbations. Dimethyl sulfoxide (DMSO) caused a marked attenuation of the 390 nm resonance, likely reflecting solvent-mediated dielectric effects or weak ligand coordination. In contrast, divalent sulfate salts (MgSO_4_ and ZnSO_4_) nearly abolished the plasmonic peak, indicative of extensive surface interaction or aggregation that disrupts collective electronic coupling (Figure 18). Sodium lauryl sulfate (SLS) maintained a sharp, undistorted resonance even at high concentrations, consistent with micellar encapsulation that preserves charge separation and inhibits aggregation (Figure 18).

To assess interactions with intracellular cofactors, ultraviolet–visible spectroscopy was recorded in the presence of nicotinamide adenine dinucleotide (NADH) and adenosine diphosphate (ADP). EVQ-218 retained a primary absorbance at ~400 nm, which red-shifted in the presence of NADH, overlapping with the nicotinamide absorption at ~260 nm (Figure 19). This spectral convergence suggests electronic coupling or charge transfer between EVQ-218 and NADH. In contrast, ADP produced a broadened spectrum with a secondary shoulder, indicative of non-redox phosphate coordination with adenine moieties. These findings imply that EVQ-218 preferentially associates with redox-active cofactors such as NADH, while interacting catalytically with nucleotide phosphates (Figure 19).

Spectral overlap analysis further revealed biomolecular specificity toward bacterial envelope components. EVQ-218 exhibited strong destructive interaction with disulfide-containing peptides. Conversely, a pronounced constructive overlap was observed with lipopolysaccharide (LPS), whose absorbance closely paralleled that of EVQ-218 near 400 nm and facilitated the dispersion of EVQ-218 (Figure 20). This alignment suggests preferential interaction with polysaccharide or lipid-rich domains within LPS for dispersion.

Together, these observations define a dual interaction paradigm for EVQ-218: electronic coupling with NADH, implicating redox-mediated intracellular activity, and high-affinity association with LPS, suggesting surface-level engagement of bacterial membranes. This multifaceted behavior provides a mechanistic framework for the antimicrobial potential of EVQ-218, bridging metabolic interference with structural disruption.

3. Discussion

The bacterial outer membrane forms a dynamic interface between the cell and its external environment, mediating both protection and molecular exchange (Figure 21) [26]. This asymmetric bilayer comprises hydrophilic head groups, fatty acyl chains, and an extensive network of lipopolysaccharides (LPS) that extend outward from the surface [27,28,29]. LPS molecules not only reinforce the membrane’s structural integrity but also serve as amphipathic scaffolds that capture, solubilize, and guide exogenous compounds toward membrane transport proteins [29].

Embedded within this matrix are specialized channels and transporters that regulate molecular influx. Porins, organized as β-barrel structures, permit the passive diffusion of small hydrophilic solutes, whereas ATP-binding cassette (ABC) transporters mediate active uptake of specific substrates through ATP hydrolysis [30,31]. Additional systems, such as the phosphotransferase system (PTS), integrate transport with substrate phosphorylation, enhancing metabolic assimilation [32]. Together, these features render the bacterial envelope both a robust barrier and a regulated gateway for antimicrobial entry.

EVQ-218 exploits these molecular entry points. Upon solubilization and dispersion by the LPS layer, EVQ-218 is guided through porins and transporter proteins into the periplasmic space and cytoplasm. Once internalized, EVQ-218 targets protein disulfide bonds, disrupting tertiary and quaternary structures and leading to protein misfolding and loss of function (Figure 21). Consistent with its known affinity for phosphate groups in ADP, EVQ-218 may also interact with the exposed nucleic acids in bacterial cytoplasm by binding to exposed phosphate backbones of DNA. This interaction is hypothesized to be predominantly electrostatic rather than catalytic in nature, based on internally conducted spectroscopic analyses of purified DNA in the presence of EVQ-218. These findings are consistent with impaired transcriptional activity and destabilization of nucleic acid integrity, collectively resulting in inhibition of essential bacterial processes and subsequent cell death.

EVQ-218 nanoparticles demonstrate a promising alternative to conventional antibiotics, combining exceptional stability with potent activity against WHO-designated priority pathogens, including the ESKAPE group, without eliciting detectable resistance. This unique behavior arises from the compound’s broad-spectrum mechanism of action, characterized by a selective affinity for sulfur- and disulfide-containing biomolecules, termed the silver–sulfur siege (S^3^). Through this mechanism, EVQ-218 disrupts essential protein structures and redox pathways across diverse bacterial taxa. Distinct from ionic silver formulations, EVQ-218 represents a stable, non-ionic silver allotrope with minimal cytotoxicity and remarkable physicochemical resilience.

From a translational perspective, these findings establish a clear path forward. Immediate next steps include in vivo evaluation to define pharmacokinetics, tissue distribution, and therapeutic windows, as well as assessment of efficacy in infection models relevant to pulmonary, device-associated, and biofilm-mediated disease. Given its nanoscale dimensions, solubility, and thermal stability, EVQ-218 is well positioned for development as an inhaled antimicrobial for respiratory infections or as a surface-active coating for medical devices to prevent bacterial colonization and biofilm formation. Together, these attributes position EVQ-218 as a compelling candidate for next-generation antimicrobial development and a potential contributor to efforts aimed at mitigating the global burden of antimicrobial resistance.

4. Materials and Methods

4.1. Bacterial Isolates

Reference strains of Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442, Klebsiella pneumoniae ATCC 4352, Enterobacter cloacae ATCC 13047, Enterococcus faecium ATCC 19434, and Acinetobacter baumannii ATCC 17978 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cultures were maintained on tryptic soy agar (TSA) and propagated overnight in Mueller–Hinton broth (MHB; MilliporeSigma, Burlington, MA, USA) at 37 °C with shaking.

4.2. Propagation and Resistance Selection

Overnight cultures were diluted to ~1.5 × 10^8^ CFU mL^−1^ (OD_600_ ≈ 0.1). Fifty microlitres of suspension were added to 5 mL of 40% cation-adjusted MHB (CAMHB) and diluted 1:10 into 48-well plates containing 500 µL of EVQ-218 at defined concentrations. Plates were incubated at 37 °C for 24 h, and visible turbidity confirmed growth.

For stable baseline propagation, overnight cultures were adjusted to 1.5 × 10^6^ CFU mL^−1^ in CAMHB and co-incubated with 0.625 µg mL^−1^ EVQ-218. Every 24 h, fresh subcultures were initiated under identical conditions for up to 30 passages (Table 3). Isolates were streaked on TSA every five passages for subsequent MIC testing. The EVQ-218 lot numbers are: MgAg240828-101-2F-22F2 and MgAg250714-302.

4.3. Minimum Inhibitory Concentration (MIC) Assays

MICs were determined following CLSI guidelines. EVQ-218 was serially diluted two-fold (0.17–22 µg mL^−1^) in double-distilled water. In 96-well plates, 100 µL of each dilution were combined with 100 µL of bacterial suspension (1.5 × 10^6^ CFU mL^−1^ in 20% CAMHB). Growth controls lacked compound; blanks contained medium only. Plates were incubated at 37 °C with orbital shaking, and optical density at 600 nm was measured every 30 min for 24 h using a Tecan Infinite 200 Pro (Tecan Group Ltd., Männedorf, Switzerland) or Thermo Fisher SkyHigh plate reader (Thermo Fisher Scientific, Waltham, MA, USA).

4.4. Time-Kill Kinetics

To accurately quantify CFU mL^−1^, the gold-standard approach for time-kinetics is performed in minimal media or buffered solution. In following these guidelines, and to prevent confounding interactions with continuous bacteria division, we maintained a minimal media concentration of 1% for optimized rate kinetics.

Bacterial suspensions (1.5 × 10^6^ CFU mL^−1^) were exposed to sub- and supra-MICs of EVQ-218 in 1% CAMHB. Samples were withdrawn every 30 min over 3 h, serially diluted, and plated on TSA. Colonies were counted after overnight incubation, and viable counts were expressed as CFU mL^−1^ relative to time 0.

4.5. UV-Vis Absorption Spectra

Three hundred microliters of pure 24 µg mL^−1^ of EVQ-218 in 18 MΩ·cm ultrapure water was incubated with the following and run on the ThermoFisher SkyHigh (Thermo Fisher Scientific, Waltham, MA, USA) UV-Abs from 200 nm to 800 nm (Table 4):

4.6. Antibiotic Susceptibility and Resistance Propagation

Tobramycin and ciprofloxacin MICs were determined using the same method. For resistance induction, isolates were serially passaged ten times in sub-MICs of each antibiotic (0.105–0.936 µg mL^−1^). MICs were assessed every five passages to confirm resistance development (Table 5).

4.7. Scanning Transmission Electron Microscopy and Elemental Mapping

Bacterial isolates were cultured overnight in TSB, pelleted at 10,000 rpm for 1 min, resuspended in 500 µL of 40 ppm EVQ-218, and incubated at 37 °C for 2 h with shaking. Cells were fixed in glutaraldehyde, embedded in EPON15 resin (Electron Microscopy Sciences (Hatfield, PA, USA), University of Utah, Salt Lake City, UT, USA), sectioned, and mounted on 300-mesh copper grids. Samples were carbon-coated (~2–4 nm) with Leica ACE600 coater (Nanofab Lab, University of Utah, Salt Lake City, UT, USA), and imaged using a JEOL 2800 STEM (JEOL Ltd., Tokyo, Japan) at 200 kV (Electron Microscopy Surface Analysis Lab, University of Utah, Salt Lake City, UT, USA). Elemental mapping was performed by energy-dispersive X-ray spectroscopy (EDS; Thermo Fisher Scientific, University of Utah, Salt Lake City, UT, USA) and analyzed with Thermo Fisher Scientific NSS Ver 3.2. Images were processed using ImageJ (Ver 1.54f) for contrast optimization.