Comparative performance of electronegative membrane filtration and automated concentrating pipette for detection of antibiotic resistance genes and microbial markers in river water samples

The target viral and bacterial concentrations in river water are essential for environmental monitoring and public health studies. Filtration-based methods are commonly employed, yet challenges arise due to recoverability and filter pore size. This study aimed to compare the performance of electronegative membrane filtration (EMF) and automated Concentrating Pipette (CP) Select (InnovaPrep) methods for quantifying antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and bacterial and viral markers in river water samples. Fifty-four river water samples were collected from upstream and downstream locations in a river in Japan. The CP Select method was modified by adding MgCl 2 and using different tips. The recovery efficiencies for total coliforms and Escherichia coli were assessed, and class 1 integron-integrase gene ( intI 1), 16S rRNA, gene encoding sulfonamide resistance ( sul 1), cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), and Escherichia coli gene ( sfmD ) were detected. CP Select showed recovery efficiencies of 45 %–63 % for total coliforms and 17 %–35 % for E. coli. The intI 1, 16S rRNA, sul 1, crAssphage, PMMoV, and sfmD concentrations using the modified CP Select method were 10.1 ± 0.5, 8.7 ± 0.2, 7.7 ± 0.2, 6.7 ± 0.2, 5.4 ± 0.2, and 3.5 ± 0.5 log 10 copies/L, respectively. Higher intI 1 and sul 1 concentrations were observed downstream, with the highest contribution percentage (22 % and 21 %) using CP Select or EMF. The modified CP Select method with 0.05 μm tips yielded more quantifiable results for all target genes and greater PMMoV concentrations ( p < 0.05). Positive correlations were found among bacterial, ARG/MGE, and viral markers (Spearman's ρ = 0.71 for 16S rRNA and sfmD , 0.88 for intI 1 and sul 1, and 0.64 for PMMoV and crAssphage). The modified CP Select method demonstrated effective recovery of bacteria and quantification of ARGs, MGEs, and microbial markers in river water. Further studies are required to validate these methods and confirm their applicability in diverse environmental contexts. • The performance of filtration-based concentration methods was evaluated. • CP Select exhibited mean recoveries of 63 % for total coliforms and 38 % for E. coli . • Modified protocols increased sample volume by ∼13 % but required 3:16 min more. • The 0.05 μm tips had higher bacterial and viral concentrations than 0.45 μm tips. • Modified CP Select settings improved the quantification of all target genes.