Nanoparticles and Extracellular Antibiotic Resistance Genes: Understanding the Role Naturally Occurring Nanoscale Materials May Play in Regulating the Environmental Rise of Antibiotic Resistance

Abstract

The use of antibiotics as the major defense to infectious disease since the early 20th century has led to the widespread proliferation of antibiotic resistance. This is one of the greatest current human health crises because it has led to decreased effectiveness of antibiotics and subsequently worldwide increases in deaths and economic costs. Mobile genetic elements coding for antibiotic resistance may be agents of spread. Among these, are extracellular antibiotic resistance genes (eARGs), which are environmentally relevant. Extracellular DNA (eDNA) in the environment typically breaks down quickly upon contact with ubiquitous nucleases. However, eDNA has been known to persist in association with naturally occurring particles. A relevant but yet unconsidered subset of particles that may alter the fate of eDNA, or eARGs, are nano-scale particles (NPs). Because NPs are ubiquitous in natural environments, increasing in incidence due to anthropogenic use and have unique properties, their impacts on eARG spread may be significant. This study was designed, therefore, to assess NP effects on eARG association, persistence, and bioavailability. The first objective was to characterize the ability of naturally occurring NP surfaces to associate eARGs. Previous studies have demonstrated association of environmental particles with eDNA, but none have considered particles on the nanoscale. For this objective, sorption isotherms were developed describing the association of linear DNA fragments isolated from widespread eARGs (blaI and nptII) with a model naturally occurring NP, a silica nanoparticle (SNP), and micron-sized kaolinite, which was tested as a comparison because its association with eDNA has already been well documented. For each isotherm, eARG fragments were added at five starting concentrations (5–40 μg/mL) to mixed batch systems with 0.25 g of particles and nuclease-free water. Sorption was quantified by the removal of DNA from solution, as detected by a Qubit fluorimeter. Isotherms were developed for eARGs of various fragment lengths (508, 680 and 861 bp), guanine-cytosine (GC) contents (34%, 47% and 54%) and both double and single stranded eARGs, to assess the impact of DNA properties on particle association. Sorption isotherms were also developed in systems with added humic acid and/or CaCl2, to assess the impact of these environmental parameters on sorption. Fourier transform infrared (FTIR) spectroscopy was performed to analyze the conformation of sorbed eARGs. Desorption of eARGs was studied by quantifying the removal of eDNA from washed and vortexed post-sorption particles. Irreversible sorption of eARGs to environmentally relevant NPs (humic acid functionalized silica nanoparticles) was demonstrated for the first time. Nano-emergent properties did not increase sorption capacity of eARGs but led to a unique compressed conformation of sorbed eARGs. The addition of humic acid, increased CaCl2 concentration and small DNA fragment size favored sorption. NPs showed a slight preference for the sorption of single-stranded DNA over double- stranded DNA. These findings suggest that NP association with eARGs may be a significant and unique environmental phenomenon. The second objective was to determine if NP-association impacted the persistence of eARGs. To test persistence, fragments of widespread eARG blaI were added at 2 μg/mL to batch systems containing nuclease-free water at pH 7.0, 0.1 M CaCl2 and 0.25 g of particles. Tested particles included humic acid functionalized silica nanoparticles (HASNPs) and kaolinite. After equilibration of eARG fragments and particles, pH was changed (between 1.0 and 11.0) or DNase I was added (at concentrations between 0.5 and 20 μg/mL). Remaining eARG fragment copies were characterized via the quantitative polymerase chain reaction (qPCR) and automated gel electrophoresis. eARGs fragments of various sizes (508 bp, 680 bp and 861 bp) and both double and single stranded eARGs were tested. Sorption capacity of DNase I to NPs was also assessed using the Bradford assay for protein detection. Overall, particles improved eARG persistence. HASNPs protected eDNA from breakage in low pH systems. Kaolinite provided full protection at all DNase I concentrations tested. Degradation of eARGs was significant in HASNP systems but was markedly decreased compared to particle-free systems at DNase I concentrations less than 10 μg/mL. DNase I sorbed significantly to HASNPs. Full protection of eARGs was observed, at DNase I concentrations less than 5 μg/mL, when HASNPs were adsorbed to DNase I. The impact of HASNPs on reducing degradation (at a low DNase I concentration of 1 μg/mL) was observed for up to four hours. Smaller and single stranded fragments, which have greater sorption capacities to HASNPs, were not better protected. This study establishes the ability of naturally occurring NPs to decrease the degradation of eARGs, either through sequestration of eDNA or inactivation of nucleases by particles. The third objective was to determine if persistent NP-associated eARGs were more bioavailable than free eARGs. eARGs were extracted from a multidrug resistant strain of B. subtilis (1A189), which has one point mutation in the promoter region of the blt gene, and exposed to non-resistant competent recipient cells of B. subtilis (1A1). This was done in systems with no particles, HASNPs or micron-sized humic acid functionalized silica particles (HASPs), which were tested to compare particle size effects. After a 90-minute incubation, cells were plated on selective media and incubated overnight at 37 C. Transconjugants were counted as colonies post-incubation. This transformation assay was repeated using five PCR-amplified double-stranded eARG fragments of extracted B. subtilis 1A189 of various sizes (191 bp, 400 bp, 906 bp, 1100 bp and 1500 bp) and one single-stranded fragment (906 bp) to test impacts of DNA properties on transformation. All fragments were chosen such that they symmetrically flank the point mutation. The transformation assay was also similarly performed using extracted DNA from antibiotic-resistant strains of B. subtilis - 1A354 (which has a mutated plasmid-borne sul gene) and 1A491 (which has multiple mutations on the chromosomal dfrA24 gene) to determine impacts of resistance gene type on transformation. When no nucleases were present, particles decreased bacterial growth and eARG bioavailability. When significant concentrations of DNase I were present (5 g/mL and higher), particles increased transformation and HASNPs had a greater enhancing effect than HASPs. eARG size and strand conformation did not significantly impact transformation rates. Only chromosomal eARGs increased transformation rates in response to particle addition, and transformation frequencies were higher for the eARG with a single mutation. This study established the ability of particles to increase the bioavailability of a variety of eARGs in nuclease-systems, and particularly demonstrates that NPs enhance bioavailability to a greater extent than micron-sized particles. The fourth objective was to develop a quantitative model to predict the ability of eARGs to transform bacteria, as was observed in experimental transformation assays. This model was designed using classical particle aggregation theory to represent the kinetics of free and particle-sorbed genes interacting with bacteria. eARG attachment to NPs was represented using partition coefficients extrapolated from experimental adsorption isotherms. For both free and particle-bound eARGs, eARG-bacteria interactions were represented as a two-step process of transport by Brownian motion, differential settling, and shear, followed by attachment. The model was calibrated by altering the unknown attachment efficiency parameter, , and validated using data from transformation assays performed for objective 3. In the validation step, the model predicted a transformant concentration value within 35% of the experimental result, indicating that the model is capable of predicting transformation frequencies with relative closeness. The model was highly sensitive to the value of the particle diameter and partition coefficient, but not to the  value. Comparisons between the model-predictions and experimental results suggests that the model overemphasizes the importance of particle size and surface area in regulating transformation. Even so, this model establishes a mechanistic framework from which simplified particle-particle interactions described by the rectilinear model can be used to describe a complex biological process and predict overall trends. Overall, this study is the first to characterize the importance of naturally occurring NPs in regulating the fate of eARGs. Because NPs can significantly associate with, enhance the persistence of and increase the bioavailability of eARGs, they may be important vectors for the spread of antibiotic resistance. The role of NPs in environmental resistance propagation should be further evaluated and integrated into resistance mitigation efforts.