Effects of subinhibitory quinolone concentrations on functionality, microbial community composition, and abundance of antibiotic resistant bacteria and qnrS in activated sludge

Wastewater treatment plants (WWTPs) are continuously exposed to sub-inhibitory concentrations of antibiotics that are thought to contribute to the spreading of antibiotic resistant bacteria and antibiotic resistance genes, which are eventually released to downstream environments through effluents. In order to understand the effects of sub-inhibitory concentrations of antibiotics on sludge microbiome and resistome, we spiked a conventional activated sludge (CAS) model system with ciprofloxacin, a common fluoroquinolone antibiotic, from 0.0001 mg/L (about twice the typical ciprofloxacin concentration observed in municipal wastewater) up to 0.1 mg/L (one order of magnitude below the clinical MIC for Enterobacteriaceae) for 151 days. The abundance of ciprofloxacin resistant bacteria and qnrS, a plasmid-associated gene that confers resistance to quinolones, in activated sludge and in effluents of control and spiked CAS reactors, showed no measurable effect of the antibiotic amendment. This was also true for the bacterial community structure and for indicators of WW treatment such as N removal efficiency. Surprisingly, temporal fluctuations in both reactors could explain the observed internal variability of these antibiotic resistance determinants better than the hypothesized antibiotic-driven selective pressure. Overall, this work shows that the core sludge microbiome in CAS systems is resilient to sub-inhibitory concentrations of ciprofloxacin at a functional, structural, and antibiotic resistance levels.