Investigating and Modeling the Regulation of Extracellular Antibiotic Resistance Gene Bioavailability by Naturally Occurring Nanoparticles

Abstract

Extracellular antibiotic resistance genes (eARGs) are widespread in the environment and can genetically transform bacteria. This work examined the role of environmentally relevant nanoparticles (NPs) in regulating eARG bioavailability. eARGs extracted from antibiotic-resistant B. subtilis were incubated with nonresistant recipient B. subtilis cells. In the mixture, particle type (either humic acid coated nanoparticles (HASNPs) or their micron-sized counterpart (HASPs)), DNase I concentration, and eARG type were systematically varied. Transformants were counted on selective media. Particles decreased bacterial growth and eARG bioavailability in systems without nuclease. When DNase I was present (≥5 μg/mL), particles increased transformation via chromosomal (but not plasmid-borne) eARGs. HASNPs increased transformation more than HASPs, indicating that the smaller nanoparticle with greater surface area per volume is more effective in increasing eARG bioavailability. These results were also modeled via particle aggregation theory, which represented eARG-bacteria interactions as transport leading to collision, followed by attachment. Using attachment efficiency as a fitting factor, the model predicted transformant concentrations within 35% of experimental data. These results confirm the ability of NPs to increase eARG bioavailability and suggest that particle aggregation theory may be a simplified and suitable framework to broadly predict eARG uptake.

Keywords: antimicrobial resistance; extracellular DNA; horizontal gene transfer; nanoparticles; particle aggregation.

Figure 1:

Schematic of natural transformation assay in which eARGs extracted from donor bacteria are amplified by PCR and used to transform nonresistant recipient bacterial cells.

Figure 2:

Bacterial counts, B(CFU/mL), observed in negative control tests for the transformation protocol (No eARGs or DNase I) with various particle types.

Figure 3:

Transformation frequencies (f) in systems with varying DNase I concentrations and either HASNPs, HASPs or no particles.

Figure 4:

Transformation frequencies (f) upon exposure to eARG fragments of (A) various sizes and (B) various strand conformations and (C) various mutated eARGs in systems with HASNPs, HASPs or no particles. * Denotes statistically significant difference between groups determined by a p value <0.05.

Figure 5:

Model-predicted concentration of transformed bacteria (T) as a function of time (t) for HASP systems (N₀=0.5 μg/mL). Star depicts experimental value of T at 90 minutes.

Figure 6:

The contribution of Brownian motion (BR), differential settling (DS) and shear forces (S) on collision frequencies (β) between bacteria and (A) particle-bound eARGs and (B) free eARGs.

Figure 7:

Sensitivity of model to (A) eARG-particle complex diameter, d_L (B) attachment efficiency between particle bound eARG and bacteria, α_LB, and (C) partition coefficient, P, during adsorption.