Gentamicin at sub-inhibitory concentrations selects for antibiotic resistance in the environment

Abstract

Antibiotics released into the environment at low (sub-inhibitory) concentrations could select for antibiotic resistance that might disseminate to the human microbiome. In this case, low-level anthropogenic sources of antibiotics would have a significant impact on human health risk. In order to provide data necessary for the evaluation of this risk, we implemented river water microcosms at both sub-inhibitory and inhibitory concentrations of gentamicin as determined previously based on bacterial growth in enriched media. Using metagenomic sequencing and qPCR/RT-qPCR, we assessed the effects of gentamicin on water bacterial communities and their resistome. A change in the composition of total and active communities, as well as a gentamicin resistance gene selection identified via mobile genetic elements, was observed during a two-day exposure. We demonstrated the effects of sub-inhibitory concentrations of gentamicin on bacterial communities and their associated resistome in microcosms (simulating in situ conditions). In addition, we established relationships between antibiotic dose and the magnitude of the community response in the environment. The scope of resistance selection under sub-inhibitory concentrations of antibiotics and the mechanisms underlying this process might provide the basis for understanding resistance dispersion and associated risks in relatively low impacted ecosystems.

Fig. 1. Changes in the inferred absolute abundance and activity of the 24 most abundant genera in river water microcosms.

A Average genus relative abundance obtained from the sequencing of the 16S rRNA gene, normalized per number of copies of the 16S rRNA gene per l of water. B Average genus relative abundance obtained from the sequencing of the 16S rRNA (cDNA), normalized per 16S rRNA copies per l of water. Obtained from non-polluted water microcosms and microcosms polluted at 10, 50 or 800 ng/ml of gentamicin after 0, 1 or 2-day exposure. n = 3.

Fig. 2. Dose-response linking sub-inhibitory gentamicin concentrations and relative abundance of aminoglycoside resistance genes and transcripts.

A Relative abundance of aac(6’) family gene copies; B relative abundance of aac(6’) family transcript copies; C relative abundance of aadA family gene copies; D relative abundance of aadA family transcript copies. Blue: 0 ng/ml of gentamicin. Orange: 10 ng/ml of gentamicin. Green: 50 ng/ml of gentamicin. Dot colour gets darker with exposure time (DO; day 0, D1: day 1; D2; day 2). aac(6’) family gene primers: qPCR efficiency = 0.99; R² linearity coefficient = 0.997. aadA family gene primers: qPCR efficiency = 1; R² linearity coefficient = 0.996. ANOVA degrees of freedom: 8. ANOVA F-values: 4.88 (aac(6’) abundance); 17.25 (aac(6’) transcription); 2.52 (aadA abundance); 0.98 (aadA transcription). n = 3.

Fig. 3. Limnohabitans MAGs containing a gentamicin resistance gene obtained from the hybrid co-assembly short and long of metagenomic sequences from water microcosms polluted at 50 ng/ml of gentamicin.

A Samples exposed to gentamicin for 0, 1 and 2 days; B Samples exposed to gentamicin for 2 days. Taxonomy shown is based on Open Reading Frames (ORFs) on contigs search against the protein reference database kaiju.