Combined Detection of Intracellular and Extracellular Antibiotic Resistance Genes in Municipal Wastewater—Sample Preparation Methods and Storage Stability

Abstract

This study addresses the need for reliable methods in wastewater-based antimicrobial resistance monitoring by comparing three sample preparation approaches for analyzing intracellular DNA (iDNA) and extracellular DNA (exDNA) in raw wastewater: a filtration-based method, a centrifugation-based method, and a combination of the two (combined). We evaluated method repeatability, operational time, usability, consumable costs, qualitative workflow characteristics, and descriptive recovery. Selected antibiotic resistance genes (ARGs) (sul1, blaOXA48 group, blaVIM1 group, vanA, tetW, QnrS1) and the mobile genetic element intI1 were quantified by digital PCR. Most target signals occurred in the iDNA-enriched fraction (>98%). Filtration and the combined method received higher usability scores than centrifugation, whereas the combined workflow required approximately 15% higher consumable costs. Storage effects were target-, fraction-, and temperature-dependent. No common holding time satisfied the complete two-sided operational acceptance criteria. Considering decreases in relative recovery alone, storage at 5 °C for up to six days did not reduce any mean recovery below its target- and fraction-specific lower limit. Storage at −20 °C produced decreases in iDNA recovery after 24 h. These findings offer practical guidance for PCR-based AMR monitoring in wastewater.