Persistence and Environmental Relevance of Extracellular Antibiotic Resistance Genes: Regulation by Nanoparticle Association

摘要

Fate of extracellular antibiotic resistance genes (eARGs)—which are widespread in various environments—is dependent on gene persistence. This work examined the previously unconsidered role of environmentally relevant nanoparticles (NPs) in regulating eARG persistence. Fragments of 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 by the quantitative polymerase chain reaction and automated gel electrophoresis. eARGs fragments of various sizes (508, 680, 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 extracellular DNA (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 with particle-free systems at DNase I concentrations 10 μg/mL. DNase I sorbed significantly to HASNPs. Full protection of eARGs was observed, at DNase I concentrations 5 μg/mL, when HASNPs were adsorbed to DNase I. The impact of HASNPs on reducing degradation (at 1 μg/mL DNase I) was observed for up to 4 h. 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 ability of NPs to increase eARG persistence may have implications for the dissemination of antibiotic resistance.