Effects of subinhibitory quinolone antibiotic concentrations on microbial communities and abundance of antibiotic resistant bacteria and genes in activated sludge wastewater treatment

Wastewater treatment plants (WWTPs) receive daily loads of sub-inhibitory concentrations of antibiotics and are believed to foster onset and spreading of antibiotic resistant bacteria and antibiotic resistance genes eventually released to downstream environments through effluents. The conventional activated sludge (CAS) is the most applied biological process for C, N, and P removal in WWTP, and the core of global wastewater treatment. In order to dissect and investigate the effects of antibiotics on the sludge microbiome and resistome, we spiked a model CAS system with ciprofloxacin, a common fluoroquinolone antibiotic, at sub-inhibitory concentrations ranging from 0.0001 mg/L (about two times 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 distribution of ciprofloxacin resistant bacteria from activated sludge and effluents monitored in the tested system in concomitance with qnrS, a plasmid-associated gene that confers resistance to quinolones, showed no measurable effect of the antibiotic with respect to the non-spiked control system. No effect was observed in the bacterial community structure and treatment performance indicators as the N removal efficiency of the system either, and natural internal fluctuations could explain the internal variability of these antibiotic resistance determinants better than the hypothesised antibiotic-driven selective pressure. Overall, this work provides evidences that the core sludge microbiome in CAS systems is resilient to sub-inhibitory concentrations of ciprofloxacin at a functional, structural, and antibiotic resistance levels.