Snapshot ARG Removal Rates across Wastewater Treatment Plants Are Not Representative Due to Diurnal Variations

Abstract

To evaluate the threat of the environmental dissemination of antibiotic resistance associated with wastewater treatment plants (WWTPs), the removal efficiency of antibiotic resistance genes (ARGs) during wastewater treatment needs to be assessed. The sample collection strategy is one factor that is often overlooked in study design and most studies on ARGs in wastewater perform grab sampling. Here, we hypothesized that wastewater sampling (i.e., grab and composite sampling) influences the observed ARG concentrations and calculated removal rates across WWTPs. We compared the removal rates calculated based on the two different sampling methods for several genes, including some clinically relevant ARGs (blaNDM-1, blaOXA-1, MCR-1, MCR-5, MCR-10, and qnrA). We conducted summer and winter 24 h sampling campaigns where grab samples were collected every 2 h from the influent, secondary effluent, and final effluent. The snapshot removal rate of each target gene calculated based on the 12 grab samples fluctuated by 0.5–1.6 log in the winter and 0.9–2.7 log in the summer, indicating diurnal variation. Overall, for each target gene, the removal rates calculated based on 24 h composite samples were approximately equal to the median of the 12 snapshot removal rates. Our study confirms the importance of using composite samples to monitor ARGs in wastewater.

On composite sampling for monitoring generic and antibiotic-resistant coliforms in irrigation ponds

“…The results of this study indicate that sampling only at the pond banks may result in an overestimation of the average ARB concentrations, while sampling only at the interior may result in an underestimation. While this study did not delve into the temporal dynamics of ARB in the studied irrigation ponds, recent research has shown that the time of day in which samples are collected may also be a significant factor governing ARB/ARG concentrations in surface waters (Lou et al, 2022;Nguyen et al, 2022) which should be accounted for in design of monitoring programs. Additionally, future work should investigate how environmental compartments for AMR in aquatic systems, such as sediments or biofilms, impact the measured concentrations in the water column.…”

Persistence of sewage-associated genetic markers in advanced and conventional treated recycled water: implications for microbial source tracking in surface waters

“…Variable persistence of microbes and their DNA can be influenced by factors such as treatment strategy utilized ( 50 ), air temperature, and elevation ( 51 ). Within a facility, the time of the day the sample is collected ( 52 ), flow rate ( 53 ), and seasonal effects ( 54 ) can all influence microbial concentrations. Post-secondary treatment stages in AWT facilities sampled here include anoxic basins and oxidation ditches, dual media deep-bed denitrification filters, activated sludge treatment in an anoxic/aerobic configuration, and a five-stage Bardenpho activated sludge process, each of which could feasibly contribute to the reduction of DNA in recycled water.…”