Silver nanoparticles are being produced globally at roughly 1 ton/day, driven by use in medical devices, food-contact surfaces, and numerous consumer products, including textiles, paints, washing machines, food containers, and cosmetic products. Widespread use is primarily due to the antimicrobial property of silver nanoparticles, which interact with cells in ways other materials cannot. However, a consequence of non-selective, indiscriminate use of silver nanoparticles is incidental concentrations of silver nanoparticles, silver ions, or silver precipitates (e.g., silver chloride, silver sulfide) in the environment. In addition to human health and environmental toxicity concerns, bacteria exposure to sublethal concentrations of these silver forms can spur the evolution of resistance, as evidenced in controlled laboratory settings and in bacteria isolated from known contaminated sites. Resistance to silver nanoparticles, like all other antibiotics and antimicrobials, has the potential to become a problem for hospitals and other industries (e.g., water treatment and distribution, food and agriculture) that rely on them for pathogen inactivation, infection prevention, and biofilm control as higher concentrations will be needed to have the same effect.
Despite the ubiquitous deployment of silver nanoparticles into consumer products, our current understanding of how to rationally design these particles for a given application in a way that ensures long-term efficacy and reduced adverse consequences is far from complete. This is because specific mechanisms of inactivation, as well as mechanisms for bacterial resistance, in relation to particle design remain unresolved. While metal resistance (to copper and silver) has been studied for decades, our understanding of resistance to silver in the form of nanoparticles is nascent. Thus, we set out to explore the mechanism of resistance to spherical silver nanoparticles capped with a polyethylene glycol polymer – one of the most commonly used types of nanoparticles in consumer products – and silver ions using E. coli as our model organism.
In our study published in Nature Nanotechnology, we combined experimental evolution, whole-population genome sequencing, and comprehensive nanomaterial synthesis and characterization to select for and evaluate mechanisms of silver nanoparticle resistance. Experimental evolution mimics how resistance develops in real-world systems, where bacteria are repeatedly exposed to sublethal doses of the antimicrobial of interest. In the laboratory, we exposed a hypermotile E. coli K-12 MG1655 (+IS1) strain to sublethal concentrations of silver nanoparticles and silver ions for twenty consecutive passages (i.e., repeated exposure). In response to the silver nanoparticles, we saw a four-fold increase in the minimum inhibitory concentration (MIC, the lowest antimicrobial concentration at which there is complete growth inhibition) occurring after eight passages, which remained stable for the twenty passages. This signifies that bacteria become resistant to silver nanoparticles, enabling them to survive at higher concentrations. We did not observe the same evolution of resistance for bacteria exposed to silver ions alone. Our findings suggest that evolution of resistance is an important design trade-off that accompanies maximizing antimicrobial activity with silver nanoparticles. Further, there is an opportunity to minimize this trade-off by manipulating the unique physicochemical parameters of the nanoparticle to express certain functionalities that can alter the resulting resistance profiles.
To probe the mechanism through which resistance evolved to the silver nanoparticles, we worked with the Microbial Genome Sequencing Centre to apply whole-population genome sequencing of bacteria isolated from multiple passages. Analysis of the genomic variants enabled us to identify the presence of an ion efflux pump mutation that likely prolongs the efflux pump activation. Resistance to silver nanoparticles is thus imparted through increased silver efflux of the original silver nanoparticles (if entered into the cell intact followed by intracellular dissolution) or silver ions (if the particles enabled localized release at the cell membrane).
Another important finding from our study is that bacterial motility - how bacteria move around and interact in their surroundings, largely imparted by the flagellum - influences silver nanoparticle resistance. We compared the resistance profiles of two E. coli strains – one displaying hyper motility (imparted by an insertion element upstream of the flhD operon) and one being non-motile with no flagella or flagellin present (imparted by the absence of flhD, the master regulator of the flagella). In contrast to the hypermotile strain, there was no conclusive indication that resistance evolved in the non-motile E. coli K-12 JW1881 (ΔflhD::kan) strain. This finding is promising for considering design of nano-enabled antimicrobials that target specific bacteria, particularly because several multidrug-resistant bacteria on the CDC’s urgent and serious threats list are non-motile (e.g., Clostridioides difficile, P. aeruginosa, Staphylococcus aureus, and various types of Acinetobacter and Streptococci).
Ongoing research is aimed at determining what form of silver is internalized and what form of silver is eliminated to tailor nanoparticle design accordingly. We speculate that silver nanoparticles are delivering a greater amount of silver into the cell and inducing intracellular toxicity, from which a direct mechanism of resistance develops to pump silver out. How the nanoparticles enhance this effect compared with the ions alone remains to be resolved. Further, it is likely that silver ions impart their toxicity extracellularly and as a result, the cell is not able to adapt to overcome the induced stress. To overcome this mechanism of resistance and maintain long-term efficacy, it is critical to focus on delivering extracellular mechanisms of inactivation through modulation of the physicochemical nanoparticle parameters, designing for high sustained ion release, or designing for a high biological fitness cost to prevent or lower the frequency of mutations arising in efflux pumps. As our study only examined one type of silver nanoparticle (one combination of size, shape, and surface chemistry) within one experimental system, additional research is also needed to inform more global conclusions that account for the wide variability of nanoparticle-dependent biological interactions, particle behavior in suspension, and underlying genetic responses.
More broadly, there is potential for rational design of silver nanoparticles to tackle global, sustainable challenges in the health care sector, water treatment and distribution, and food and agriculture. Our work underlines the need for developing and executing a sustainable action plan that limits the ubiquitous use of silver nanoparticles, reserving their use for those applications necessitating antimicrobial functions. This approach will enable long-term use for our next-generation of nano-enabled antimicrobial agents.