Repeated dose (28-day) administration of silver nanoparticles of varied size and coating does not significantly alter the indigenous murine gut microbiome

Abstract

Silver nanoparticles (AgNPs) have been used as antimicrobials in a number of applications, including topical wound dressings and coatings for consumer products and biomedical devices. Ingestion is a relevant route of exposure for AgNPs, whether occurring unintentionally via Ag dissolution from consumer products, or intentionally from dietary supplements. AgNP have also been proposed as substitutes for antibiotics in animal feeds. While oral antibiotics are known to have significant effects on gut bacteria, the antimicrobial effects of ingested AgNPs on the indigenous microbiome or on gut pathogens are unknown. In addition, AgNP size and coating have been postulated as significantly influential towards their biochemical properties and the influence of these properties on antimicrobial efficacy is unknown. We evaluated murine gut microbial communities using culture-independent sequencing of 16S rRNA gene fragments following 28 days of repeated oral dosing of well-characterized AgNPs of two different sizes (20 and 110 nm) and coatings (PVP and Citrate). Irrespective of size or coating, oral administration of AgNPs at 10 mg/kg body weight/day did not alter the membership, structure or diversity of the murine gut microbiome. Thus, in contrast to effects of broad-spectrum antibiotics, repeat dosing of AgNP, at doses equivalent to 2000 times the oral reference dose and 100-400 times the effective in vitro anti-microbial concentration, does not affect the indigenous murine gut microbiome.

Keywords: Antibiotics; in vivo; metastats; microbiome; mothur; mouse; nanomaterials; pyrosequencing; toxicology.

Figure 1

Relative abundance of bacterial phyla from mouse cecal contents after 28 days of oral gavage with 10 mg/kg bw/day of 110 nm PVP-coated AgNP in comparison to water and silver acetate. A. Mean % relative abundance of all phyla for mice from each group, with mean percentage of each phylum indicated. For all groups, Bacteroidetes was the major phylum present, followed by Firmicutes and the murine-specific phylum Deferribacteres. Other phyla (combined) were present at <4% in each group and consisted of unclassified phyla (most abundant, 1–2%), followed by Actinobacteria, Proteobacteria, Tenericutes, Verrucomicrobia, and TM7, each present at <1%. B. Median (horizontal bar), interquartile range (box) and minimum/maximum for major phyla in each group. There were no significant differences between groups with respect to microbial community composition and structure at the phylum level (ANOVA, p=0.861).

Figure 2

Cecal microbial communities in animals gavaged with 110 nm AgNP-PVP, AgOAc, or water for 28 days. Data are depicted as a principal components analysis (PCoA) plot representing the Yue Clayton (ØYC) distance metric. Microbial communities were not significantly different between groups. (AMOVA, p=0.111).

Figure 3

Relative abundance of bacterial phyla from mouse cecal contents after 28 days of oral gavage with 10 mg/kg bw/day of 110 nm, 20 nm citrate-coated, or 20 nm PVP-coated AgNP in comparison to 28 days oral gavage with water or 12 days of antibiotic (cefoperazone) administration in the drinking water. A. Mean % relative abundance of predominant phyla for mice from each group. All AgNP and water- dosed groups had a preponderance of Bacteroidetes and Firmicutes, with lesser numbers of Deferribacteres. Other phyla (combined) were present at <2% in each group and consisted of unclassified phyla, Actinobacteria, Proteobacteria, Tenericutes, Verrucomicrobia, and TM7. B. Median (horizontal bar), interquartile range (box), and minimum/maximum for major phyla identified in all groups. Only the antibiotics-treated group had significant differences from other groups, consisting of increased Firmicutes and decreased Bacteroidetes (ANOVA, Tukey’s multiple comparisons test, p<0.0001). N=5–6 for water or AgNP groups (see text). Only two points are shown for the antibiotics group since only two animals had sufficient recovery of gut bacterial DNA for sequencing remaining after antibiotic dosing, despite the 24 hour washout period.

Figure 4

Cecal microbial community differences between groups gavaged with AgNP of varying size and coating. Data are depicted as a PCoA plot representing the ØYC distance metric. Microbial communities were not significantly different between groups. (AMOVA, p=0.114).