Comparison of concentration and extraction workflows for qPCR quantification of intI1 and vanA in untreated wastewater

Yawen Liu; Wendy Smith; Metasebia Gebrewold; Xinhong Wang; Stuart L Simpson; Aaron Bivins; Warish Ahmed

doi:10.1016/j.scitotenv.2023.166442

Comparison of concentration and extraction workflows for qPCR quantification of intI1 and vanA in untreated wastewater

Sci Total Environ. 2023 Dec 10:903:166442. doi: 10.1016/j.scitotenv.2023.166442. Epub 2023 Aug 19.

Authors

Yawen Liu¹, Wendy Smith², Metasebia Gebrewold², Xinhong Wang³, Stuart L Simpson⁴, Aaron Bivins⁵, Warish Ahmed⁶

Affiliations

¹ State Key Laboratory of Marine Environmental Science, College of the Environment & Ecology, Xiamen University, Xiamen 361102, China; CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia.
² CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia.
³ State Key Laboratory of Marine Environmental Science, College of the Environment & Ecology, Xiamen University, Xiamen 361102, China.
⁴ CSIRO Environment, Lucas Heights, NSW 2234, Australia.
⁵ Department of Civil & Environmental Engineering, Louisiana State University, Baton Rouge, LA 70809, USA.
⁶ CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia. Electronic address: Warish.Ahmed@csiro.au.

PMID: 37604373
DOI: 10.1016/j.scitotenv.2023.166442

Abstract

Quantitative polymerase chain reaction (qPCR) measurement of antibiotic resistance genes (ARGs) in untreated municipal wastewater may prove useful in combating the antimicrobial resistance crisis. However, harmonizing and optimizing qPCR-based workflows is essential to facilitate comparisons across studies, and includes achieving highly-effective ARG capture through efficient concentration and extraction procedures. In the current study, combinations of sample volume, membrane types and DNA extraction kits within filtration and centrifugation-based workflows were used to quantify 16S ribosomal RNA (16S rRNA), class 1 integron-integrase gene (intI1) and an ARG encoding resistance to vancomycin (vanA) in untreated wastewater sampled from three wastewater treatment plants (WWTPs). Highly abundant 16S rRNA and intI1 were detected in 100 % of samples from all three WWTPs using both 2 and 20 mL sample volumes, while lower prevalence vanA was only detected when using the 20 mL volume. When filtering 2 mL of wastewater, workflows with 0.20-/0.40-μm polycarbonate (PC) membranes generally yielded greater concentrations of the three targets than workflows with 0.22-/0.45-μm mixed cellulose ester (MCE) membranes. The improved performance was diminished when the sample volume was increased to 20 mL. Consistently greater concentrations of 16S rRNA, intI1 and vanA were yielded by filtration-based workflows using PC membranes combined with a DNeasy PowerWater (DPW) Kit, regardless of the sample volume used, and centrifugation-based workflows with DNeasy Blood & Tissue Kit for 2-mL wastewater extractions. Within the filtration-based workflows, the DPW kit yielded more detection and quantifiable results for less abundant vanA than the DNeasy PowerSoil Pro Kit and FastDNA™ SPIN Kit for Soil. These findings indicate that the performance of qPCR-based workflows for surveillance of ARGs in wastewater varies across targets, sample volumes, concentration methods and extraction kits. Workflows must be carefully considered and validated considering the target ARGs to be monitored.

Keywords: Antibiotic resistance genes (ARGs); Concentration and extraction workflow; Wastewater surveillance; qPCR.